Nitric oxide donors and uses thereof

ABSTRACT

Disclosed are novel NO-donating compounds, designed such that when NO is released from the compound a residue which is a naturally occurring metabolite is formed, and thus a development of tolerance to the compounds upon repetitive administration is prevented or decreased. Also disclosed are methods of preparing such NO-donating compounds, pharmaceutical compositions and medical devices containing such compounds and methods utilizing such compounds in the treatment of various medical conditions.

RELATED APPLICATIONS

This application is a National Phase Application of PCT Application No.PCT/IL2005/000481 having International Filing Date of May 5, 2005, whichclaims the benefit of U.S. Provisional Patent Application No.60/567,824, filed on May 5, 2004, and U.S. Provisional PatentApplication No. 60/651,619, filed on Feb. 11, 2005. The contents of eachof the above Applications are all incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to novel, non-tolerance inducing,NO-donating compounds and their use in the treatment of variousdisorders and diseases such as, for example, cardiovascular diseases,inflammation, tumor suppression and psychiatric and neurologicaldiseases.

Nitric oxide (NO) mediates multiple physiological and pathophysiologicalprocesses in the cardiovascular and neurological systems.

Biological NO is synthesized by the enzyme nitric oxide synthase (NOS)that generates NO from L-arginine. This enzyme exists in three differentforms (referred to as isoforms), NOS-1, NOS-2 and NOS-3. Each isoformgenerates NO under different conditions. NOS-1 is the neural isoform(also known as the brain isoform) and is a key component in synaptictransmission. NOS-2 (also known as inducible NOS is responsible forgenerating high concentrations of NO (100 to 1000 folds higher then thenormal NO biological concentration), typically in response to thepresence of bacteria. NOS-2 is produced by macrophages and isresponsible for their effects to repair injury and warding offinfections. NOS-3 (also known as endothelial NOS or eNOS) is found inendothelial cells lining the inner surface of all blood vessels andlymph ducts. eNOS is activated by the pulsatile flow of blood throughvessels, which exerts “shear stress” on the membrane of the endothelialcells. The NO generated by eNOS is responsibly for maintaining thediameter of blood vessels, to thereby maintain an optimal level oftissues perfusion, as well as for the growth of new blood vessels(angiogenesis).

Pharmacological compounds that release NO (also known as NO-donors) havebeen useful tools for evaluating the pivotal role of NO in physiologyand therapeutics. These agents constitute two broad classes ofcompounds, those that release NO or one of its redox congenersspontaneously, and those that require enzymatic metabolism to generateNO. Several commonly used cardiovascular drugs exert their beneficialaction, in part, by modulating the NO pathway.

Dysfunction of the normally protective endothelium is found in severalcardiovascular diseases, including atherosclerosis, hypertension, heartfailure, coronary heart disease, arterial thrombotic disorders andstroke. Endothelial dysfunction leads to nitric oxide (NO) deficiency,which has been implicated in the underlying pathobiology of many ofthese disorders (NO insufficiency states) [Loscalzo J. and Vita J.,Nitric Oxide and the Cardiovascular System. Totawa, N.J.: Humana Press;2000]. NO insufficiency may reflect an absolute deficit of NO(synthesis), impaired availability of bioactive NO, or enhanced NOinactivation. Whatever its biochemical basis, NO insufficiency limitsNO-mediated signal transduction of normal or protective physiologicalprocesses. In light of this pathobiology, replacement or augmentation ofendogenous NO by exogenously administered NO donors has provided thefoundation for a broad field of pharmacotherapeutics in cardiovascularand neurological medicine.

The beneficial effects of nitric oxide (NO) as a therapeutic agent ingeneral, and as a blood vessel dilator (vasodilator) in particular, wasfirst observed in 1857, and demonstrated by the therapeutic activity ofa family of compounds, known as nitrovasodilators, that have been usedpurposely for almost 150 years.

While NO was originally described as a potent vasodilator [ISIS-4,Lancet. 1995; 345: 669-685 and Brunton T L, Lancet. 1867; 2: 97-98], itis now also recognized as a protecting agent against thrombosis andatherogenesis through inhibition of monocyte and platelet adhesion[Murrell W, Lancet. 1879; 1: 80-81, 11-15, 151-152, 224-227, 642-646],platelet aggregation [Chung S-J et al., J Pharmacol Exp Ther. 1990; 253:614-619] and smooth muscle cell proliferation [McGuire J J et al.,Biochem Pharmacol. 1998; 56: 881-893].

Dysfunction in NO synthesis has been implicated as a major contributoryfactor in development of a wide range of cardiovascular diseasesincluding hypertension [Kurz M A et al., Biochem J. 1993; 292: 545-550and Needleman P et al., J. Pharmacol Exp Ther. 1973; 187: 324-331],coronary artery disease and heart failure [Loscalzo J, J Clin Invest.1985; 76: 703-708 and Munzel T et al., J Clin Invest. 1995; 95:187-194]. The detrimental effects of reduced NO synthesis, as a resultof enzyme dysfunction or endothelial damage, are often exacerbated incardiovascular disease by increased generation of oxygen free radicalswhich rapidly inactivate NO [Munzel T et al., J Clin Invest. 1996; 98:1465-1470] forming cytotoxic peroxynitrite and, ultimately, inactivenitrate. Thus, delivery of supplementary NO to areas of the vasculaturewhere the protective effects of endogenous NO have been adverselyaffected is an attractive therapeutic option.

It is now well established that NO is an important bio-regulator,involved not only in blood clotting and blood pressure, but also in thecontrol of neurotransmission, and possibly the destruction of canceroustumor cells [Evig, C B. et al., Nitric Oxide, 2004, 10(3), 119-29]. NOwas found to affect neurotransmission, both directly and indirectly. NOis know to affect the cGMP level and hence promotes phosphorylation ofion channels, especially potassium channels, which are necessary fornormal transmission of nerve signals. As it affects the blood flow, NOfurther promotes the transportation of oxygen and glucose to nervecells, and thus promotes ATP production and hence potassium/sodiumhomeostasis which is essential for neurotransmission. Moreover, recentstudies have linked NO to psychiatric and neurological diseases, andsuggest the augmentation of brain NO-levels as a treatment of braindiseases characterized by excessive activity of brain dopamine systemsand/or nitric oxide systems (Brain Research Bulletin, 1996, Vol. 40, No.2, pp. 121-127, Molecular and Chemical Neuropathology, 1996, Vol. 27,Humana Press, Inc. 1044-7393/96/2703-0275 and Schizophrenia Research,1995, 15(1, 2): 65).

While NO is a gas, it may be directly administered by inhalation.However, although this administration route is used in cases whereimproved patient oxygenation is required, as, for example, in pulmonaryhypertension (high blood pressure in the lungs) and in patients withsickle cell anemia, such direct administration of the NO active form maynot reach the target organ and/or biological system, and is oftentimesassociated with both biochemical and medical complications, including,for example, methemoglobinemia and direct pulmonary injury.

In a search for alternative routes for administering NO, it was foundthat NO may be delivered and generated in situ by means of prodrugs.These prodrugs are known as NO-donors, which are metabolized by means ofan enzymatic mechanism so as to generate or release active NO.

NO-donors, which are also referred to interchangeably, herein and in theart, as NO prodrugs or NO-donating agents) are pharmacologically activesubstances that spontaneously release, or are metabolized to, NO or itsredox congeners.

Organic nitrate esters represent a class of NO-donating agents used incardiovascular therapeutics since the nineteenth century. Thepreliminary reports of the clinical use of organic nitrates and nitriteswere derived from the work of Brunton [Lancet. 1867; 2: 97-98] in 1867and the seminal work of Murrell [Lancet. 1879; 1: 80-81, 11-15, 151-152,224-227, 642-646] in 1879, which showed the clear benefits ofnitroglycerin in the treatment of angina pectoris.

Additional examples of organic nitrate and nitrite esters NO-donors thatact as nitrovasodilators include erythrityl tetranitrate,pentaerythritol tetranitrate, amyl nitrite, isosorbide dinitrate,isosorbide 5-mononitrate, and nicorandil. These compounds were found tohave direct vasoactive effects and have been used for many years totreat ischemic heart disease, heart failure, hypertension and othercardiovascular diseases [Gruetter C A et al., J Cyclic Nucleotide Res.1979; 5: 211-224, J Pharmacol Exp Ther. 1980; 214: 9-15, J Pharmacol ExpTher. 1981; 219: 181-186 and ISIS-4, Lancet. 1995; 345: 669-685]. Theprincipal action of these compounds involves vasorelaxation, mediated byguanylyl cyclase activation and by direct inhibition of nonspecificcation channels in vascular smooth muscle cells (VSMCs). As such, theseagents represent the prototypical form of NO-replacement therapy.

The mechanism of action by which organic nitrate esters such asnitroglycerin generate bioactive NO typically involves enzymaticmetabolism. Studies conducted in this respect suggested thatnitroglycerin (glyceryl trinitrate; GTN) induces vasorelaxation bygenerating NO or a related S-nitrosothiol (SNO), which is formed bydirect interactions of GTN with low-molecular-weight thiols [28],through an enzymatic system that is located within microsomal membranes,has an estimated apparent molecular mass of 160 kDa, and manifestsenhanced activity in the presence of reducing equivalents, especiallythiols [Chung S-J et al., J Pharmacol Exp Ther. 1990; 253: 614-619],which are known to potentiate the action of organic nitrate esters[Napoli C et al., Nitric Oxide. 2001; 5: 88-97 and Loscalzo J. et al., JClin Invest. 1985; 76: 703-708].

Thus, it was found that NO and SNO activate the soluble target enzymeguanylyl cyclase (sGC), increasing tissue levels of the second messengercGMP. A cGMP-dependent protein kinase I (cGK-I) mediates vasorelaxationby phosphorylating proteins that regulate intracellular Ca2+ levels[Lincoln, T. M. et al., J. Appl. Physiol, 2001. 91:1421-1430]. However,it was further found that nitroglycerin could also dilate blood vesselsthrough a cgMP-independent pathway [Chen, Z. et al., Proc. Natl. Acad.Sci. U.S.A. 2002, 99:8306-8311].

Other candidate enzymes that were suggested as being involved in NOmetabolism includes glutathione S-transferases [Lau, D. T. et al.,Pharm. Res., 1992, 9:1460-1464], the cytochrome P-450 system inconjunction with NADPH and glutathione-S-transferase activities [McGuireJ J, et al., Biochem Pharmacol. 1998; 56: 881-893, Kurz M A et al.,Biochem J. 1993; 292: 545-550, McDonald, B. J. et al., Biochem.Pharmacol., 1993, 45:268-270], xanthine oxido reductase [O'Byrne, S. etal., J. Pharmacol. Exp. Ther., 2000, 292:326-330], and mitochondrialaldehyde dehydrogenase (ALDH-2) [Chen, Z. et al., Proc. Natl. Acad. Sci.USA 2002, 99: 8306-8311].

However, while the beneficial effects of administering NO-donors havebeen widely recognized, treatment with conventional nitratepreparations, as those described hereinabove, is typically limited bytheir therapeutic bioavailability half-life, lack of selectivity,systemic absorption accompanied by potentially adverse hemodynamiceffects, and drug tolerance, with the latter being with the presentlymost limiting feature associated with administration of NO-donors[Ignarro L J. et al., J Cardiovasc Pharmacol. 1999; 34: 879-886, KojdaG. et al., Cardiovasc Res. 1999; 43: 562-571, Loscalzo J. et al., HumanaPress; 2000, Loscalzo J. et al., Circ Res. 2001; 88: 756-762, LoscalzoJ., Circulation. 2000; 101: 2126-2129 and Napoli C. et al., NitricOxide. 2001; 5: 88-97]. The inadequacies in current NO-donor prodrugshave limited their use to only short-term management of angina pectorisand acute heart failure.

Since drug-tolerance presently presents the most challenging limit forthe clinical use of organic nitrite and nitrate esters, efforts havebeen made to study this phenomenon. Initially, it was hypothesized thattolerance was caused by abnormalities in the nitrate biotransformationprocess (also referred to in the art as mechanism-based or classicaltolerance), but recent investigations associated it with increasedangiotensin II-dependent vascular production of superoxide anion fromNAD(P)H oxidase and endothelial NO synthase (eNOS) [Munzel T. et al., JClin Invest. 1995; 95: 187-194 and Munzel T, Kurz S, Rajagopalan S,Thoenes M, Berrington W R, Thompson J A, Freeman B. et al., J ClinInvest. 1996; 98: 1465-1470]. The superoxide anion generated by theseenzymes reacts with NO derived from the NO donor to form peroxynitrite(OONO⁻), as indicated by the finding of increased urinary3-nitrotyrosine in nitrate-tolerant patients [Skatchkov M. et al., JCardiovasc Pharmacol Ther. 1997; 2: 85-96]. Moreover, it was found thatnitrate tolerance is also associated with cross-tolerance toendothelium-derived NO [Molina C R. et al., J Cardiovasc Pharmacol.1987; 10: 371-378], both by the oxidative inactivation of thisendogenous NO to peroxynitrite and by the “uncoupling” of eNOS activity[Munzel T. et al., Circ Res. 2000; 86: E7-E12].

The mechanisms underlying this time- and dose-dependent tolerancephenomenon are probably multifactorial and may involve neurohormonalcounter regulatory mechanisms (also known as pseudo tolerance) [Gori, T.et al., Circulation. 2002, 106:2404-2408], increases in activity of thephosphodiesterase 1A1 [Kim, D. et al., Circulation. 2001,104:2338-2343], desensitization of the sGC [Artz, J. D. et al., J. Biol.Chem. 2002, 277:18253-18256], increases in production of reactive oxygenspecies (ROS) [Munzel, T. et al., J. Clin. Invest. 1995, 95:187-194],and impairment of GTN biotransformation (also known as mechanism-basedor classical tolerance) [Chen, Z. et al., Proc. Natl. Acad. Sci. USA2002, 99:8306-8311].

Most recently, Chen et al. [Proc. Natl. Acad. Sci. USA 2002,99:8306-8311] demonstrated that the biotransformation of GTN isprimarily induced by ALDH-2, which catalyzes the conversion of GTN to1,2-glyceryl dinitrate (1,2-GDN) and nitrite within mitochondria. Thestudy of Chen et al. demonstrated that inhibitors of ALDH-2 blocked thevasorelaxation by GTN, which is dependent on cGMP (cGMP-independentrelaxation was still evident), both in vitro and in vivo, andfurthermore, that treatment of vascular tissue with high concentrationsof GTN resulted in both inhibition of ALDH-2 and a shift in the GTN doseresponse relationship. Thus, it appears that inhibition of ALDH-2 alsounderlies classical mechanism-based tolerance in vitro. Chen et al.speculated that build up of GTN and/or NO by-products in mitochondriamay lead to mitochondrial damage and uncoupling of respiration, wherebyincreased production of superoxide and other ROS would in turn oxidizecritical thiols, including active-site thiols in ALDH-2 [Steinmetz, C.G. et al., Structure. 1997, 5:701-711], further attenuatingGTN-biotransformation. Superoxide also inactivates endothelium-dependentvasodilators (thereby reducing cGK-I activity). Thus, mitochondrialproduction of ROS would promote both mechanism-based tolerance andcross-tolerance.

While the extent to which ALDH-2 contributes to GTN tolerance (impairedrelaxation to GTN) and cross-tolerance (e.g., impairedendothelium-dependent relaxation) in vivo remains to be elucidated,these studies clearly indicate that the present use of organic nitratesas NO-donors is highly limited.

In order to repress, reverse or prevent nitrate tolerance, severalagents and metabolites, such as low molecular weight thiols, ascorbate,L-arginine, tetrahydrobiopterin, hydralazine, ACE (angiotensinconverting enzyme) inhibitors, and folate, have been used [Juggi, J S.et al., Can J Cardiol, 1991, 9(7), 419-25].

As an alternative treatment, novel NO-donating drugs which may offerselective effects, a prolonged half-life, and a reduced incidence ofdrug tolerance are currently in various developmental stages. Amongthese are diazeniumdiolates, known as “NONOates” (1-substituteddiazen-1-ium-1,2-diolates, e.g., DETA NONOate) [Keefer L K. et al.,Methods Enzymol. 1996, 268, pp. 281-93], S-nitrosothiols (e.g., SNAP)[Ng E S, Kubes P, Can J Physiol Pharmacol. 2003, 81(8), pp. 759-64] andmesoionic oxatriazoles (e.g., GEA3162 or1,2,3,4-oxatriazolium-5-amino-3-(3,4-dichlorophenyl)-chloride) [Karup G.et al., Pol J Pharmacol. 1994, 46(6), pp. 541-52]. However, heretoforethese compounds are still in pre-clinical phases and are mostly used asbiochemical and pharmacological tools

In view of the limitations associated with utilizing the presently knownNO-donors in modern armamentarium, there is a widely recognized needfor, and it would be highly advantageous to have novel NO-donatingcompounds devoid of the above limitations.

SUMMARY OF THE INVENTION

According to one aspect of the present invention described hereinbelow,there is provided an NO-donating compound, and/or a pharmaceuticallyacceptable salt thereof, which includes an NO-releasing group and achemical moiety being covalently attached to the NO-releasing group,such that when NO is released from the compound, a residue which is anaturally occurring metabolite is formed, thereby preventing ordecreasing a development of tolerance to the NO-donating compound uponrepetitive administration thereof.

According to further features in preferred embodiments of the inventiondescribed below, the NO-releasing group is selected from the groupconsisting of a —ONO₂ group, a —SNO group, a diazeniumdiolate and amesoionic oxatriazole.

According to still further features in the described preferredembodiments the NO-donating compound further includes a bioactive agentresidue, as described hereinbelow, covalently attached to the chemicalmoiety.

According to still further features in the described preferredembodiments the bioactive agent residue is attached to the chemicalmoiety via a biocleavable moiety, as described hereinbelow.

According to still further features in the described preferredembodiments the naturally occurring metabolite is a thiamine metabolite.

According to still further features in the described preferredembodiments the chemical moiety includes a substituted or unsubstitutedthiazole ring.

According to still further features in the described preferredembodiments the NO-donating compound has the general formula I:

wherein:

A is selected from the group consisting of alkenyl, alkoxy, alkyl,alkynyl, amine, amine-oxide, aryl, aryloxy, azo, borate, C-amide,carbonyl, C-carboxylate, C-thiocarboxylate, cycloalkyl, diazo,disulfide, guanidine, guanyl, haloalkyl, heteroalicyclic, heteroaryl,hydrazine, N-amide, N-carbamate, N-dithiocarbamate, nitro,N-sulfonamide, N-thiocarbamate, O-carbamate, O-carboxylate,O-thiocarbamate, O-thiocarboxylate, oxime, oxygen, sulfur, peroxo,phosphate, phosphine-oxide, phosphine-sulfide, phosphinyl, phosphite,phosphonate, pyrophosphate, S-dithiocarbamate, silaza, silicate, siloxy,silyl, S-sulfonamide, sulfate, sulfite, sulfonate, sulfoxide, sulfur,thioalkoxy, thioaryloxy, thiocarbonyl, thiophosphate, thiosulfate,thiosulfite, thiourea, triphosphate, urea, a biocleavable moiety and anycombination thereof, or absent;

X is selected from the group consisting of acyl-halide, alkenyl, alkoxy,alkyl, alkynyl, amine, amine-oxide, aryl, aryloxy, azo, borate, C-amide,carbonyl, C-carboxylate, C-thiocarboxylate, cyano, cycloalkyl, diazo,disulfide, guanidine, guanyl, halide, haloalkyl, heteroalicyclic,heteroaryl, hydrazine, hydrogen, hydroxy, N-amide, N-carbamate,N-dithiocarbamate, nitro, N-sulfonamide, N-thiocarbamate, O-carbamate,O-carboxylate, O-thiocarbamate, O-thiocarboxylate, oxime, peroxo,phosphate, phosphine-oxide, phosphine-sulfide, phosphinyl, phosphite,phosphonate, pyrophosphate, S-dithiocarbamate, silaza, silicate, siloxy,silyl, S-sulfonamide, sulfate, sulfite, sulfonate, sulfoxide,thioalkoxy, thioaryloxy, thiocarbonyl, thiohydroxy, thiophosphate,thiosulfate, thiosulfite, thiourea, triphosphate, urea, a bioactiveagent residue as described herein, a moiety containing one or moreNO-releasing group as described herein, a substituted or unsubstitutedthiazole and any combination thereof;

B is selected from the group consisting of a saturated or unsaturated,substituted or unsubstituted alkylene chain having 1-20 carbon atoms,and a saturated or unsaturated, substituted or unsubstituted alkylenechain having 1-20 carbon atoms interrupted by one or more heteroatom,whereby the heteroatom or heteroatoms include oxygen, sulfur, nitrogen,phosphor, silicon and any combination thereof;

Y is the NO-releasing group; and

Z is selected from the group consisting of hydrogen, alkyl, alkenyl,alkynyl, amine, cycloalkyl, heteroalicyclic, aryl, heteroaryl, halide,haloalkyl, hydroxy, thiohydroxy, alkoxy, thioalkoxy, aryloxy andthioaryloxy.

According to still further features in the described preferredembodiments the bioactive agent residue is selected from the groupconsisting of a fatty acid residue, a metabolite residue, a carbohydrateresidue, an amino acid residue, a peptide residue, a protein residue, ahydroxamic acid residue, a nicotinic acid residue, a nicotinamideresidue, a carnitine residue, a co-enzyme residue, a beta caroteneresidue, a bromelain residue, a steroidal anti-inflammatory agentresidue, a non-steroidal anti-inflammatory drug residue, ananti-psychotic agent residue, an anti-thrombogenic agent residue, ananti-platelet agent residue, an anti-coagulant residue, an anti-diabeticagent residue, a growth factor residue, a statin residue, a toxinresidue, an antimicrobial agent residue, an analgesic residue, ananti-metabolic agent residue, a vasoactive agent residue, a vasodilatoragent residue, a prostaglandin residue, a hormone residue, a thrombininhibitor residue, an enzyme residue, an oligonucleotide residue, anucleic acid residue, an antisense residue, a protein residue, anantibody residue, an antigen residue, a vitamin residue, animmunoglobulin residue, a cytokine residue, a cardiovascular agentresidue, a chemotherapeutic agent residue, an antioxidant residue, aphospholipid residue, an anti-proliferative agent residue, a heparinresidue, and any combination thereof.

According to still further features in the described preferredembodiments the bioactive agent residue is a non-steroidalanti-inflammatory drug residue, whereby the non-steroidalanti-inflammatory drug is, for example, aspirin, celecoxib, diclofenac,diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin,ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone,naproxen, oxaprozin, oxyphenbutazone, phenylbutazone, piroxicam,rofecoxib sulindac and tolmetin.

According to still further features in the described preferredembodiments the bioactive agent residue is an anti-diabetic agentresidue, whereby the anti-diabetic agent can be, for example, acarbose,acetohexamide, chlorpropamide, glimepiride, glipizide, glyburide, lipoicacid, meglitol, metformin, miglitol, nateglinide, pioglitazone,repaglinide, rosiglitazone, tolazamide, tolbutamide and troglitazone.

According to still further features in the described preferredembodiments the NO-releasing group (Y in Formula I above) is selectedfrom the group consisting of a —ONO₂ group, a —SNO group, adiazeniumdiolate and a mesoionic oxatriazole, and is preferably a —ONO₂group.

According to still further features in the described preferredembodiments Z in Formula I above is an alkyl, preferably methyl.

According to still further features in the described preferredembodiments B in Formula I above comprises an ethylene chain.

According to still further features in the described preferredembodiments B in Formula I above is selected from the group consistingof —CH₂—CH₂—O—CH₂—, —CH₂—CH₂—NH—CH₂— and —CH₂—CH₂—S—CH₂—.

According to still further features in the described preferredembodiments X in Formula I above is hydrogen or an alkyl such as, forexample, methyl, ethyl and isopropyl.

According to still further features in the described preferredembodiments X in Formula I above is a haloalkyl, such as, for example,trifluoromethyl.

According to still further features in the described preferredembodiments X in Formula I above is aryl, such as, for example, asubstituted or unsubstituted phenyl and a substituted or unsubstitutednaphthalenyl.

According to still further features in the described preferredembodiments the substituted phenyl is, for example,4-trifluoromethylphenyl and pentafluorophenyl.

According to further features in preferred embodiments of the inventionX in Formula I above is a heteroaryl, such as, for example,pyridin-3-yl.

According to still further features in the described preferredembodiments X in Formula I above is a heteroalicyclic, such as, forexample, piperidine-4-yl.

According to still further features in the described preferredembodiments X in Formula I above is amine, such as, for example, —NH₂and —N(CH₃)₂.

According to still further features in the described preferredembodiments X in Formula I above is an alkoxy, such as, for example,methoxy.

According to still further features in the described preferredembodiments X in Formula I above is a moiety containing one or moreNO-releasing group(s), such as, for example, 1-nitrooxy-ethyl,[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-diazene,4-methyl-5-(2-nitrooxy-ethyl)-thiazole and2-butyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole.

According to still further features in the described preferredembodiments X in Formula I above is a non-steroidal anti-inflammatorydrug residue, such as, for example, an aspirin residue, an ibuprofenresidue and a naproxen residue.

According to still further features in the described preferredembodiments X in Formula I above is an anti-diabetic agent residue, suchas, for example, a lipoic acid residue.

According to still further features in the described preferredembodiments A in Formula I above is a biocleavable moiety and X is abioactive agent, as described herein.

According to still further features in the described preferredembodiments the biocleavable moiety can be, for example, amide,carboxylate, carbonate, carbamate, phosphate, hydrazide, thiohydrazide,disulfide, epoxide, peroxo and methyleneamine.

Exemplary NO-donating compounds according to this and other aspects ofthe present invention are set forth in Tables 1 and 2 hereinbelow.

According to another aspect of the present invention there is provided apharmaceutical composition, which comprises, as an active ingredient, anNO-donating compound as described hereinabove, and a pharmaceuticallyacceptable carrier.

According to still further features in the described preferredembodiments the pharmaceutical composition is packaged in a packagingmaterial and identified in print, in or on the packaging material, foruse in the treatment of a medical condition in which modulating an NOlevel is beneficial. Preferably modulating the NO level includeselevating the NO level.

According to still further features in the described preferredembodiments the medical condition in which modulating an NO level isbeneficial is selected from the group consisting of a cardiovasculardisease or disorder, a gastrointestinal disease or disorder, aninflammatory disease or disorder, a respiratory disease or disorder, acentral nervous system disease or disorder, a neurodegenerative diseaseor disorder, a psychiatric disease or disorder, a bloodpressure-associated disease or disorder, a coronary artery disease ordisorder, atherosclerosis, a cholesterol level-associated disease ordisorder, an arterial thrombotic disease or disorder, a heart failure, astroke, a septic shock, a NSAID-induced gastric disease or disorder, aninflammatory bowel disease or disorder, an ischemic renal disease ordisorder, a peptic ulcer, diabetes, pulmonary hypertension, sickle cellanemia, asthma, a chronic obstructive pulmonary disease or disorder,dementia, epilepsy, a neuroinflammatory disease or disorder, trauma,multiple sclerosis, an erectile dysfunction, a male and female sexualdysfunction and an age-related disease or disorder.

According to yet another aspect of the present invention there isprovided a method of treating or preventing a medical condition in whichmodulating an NO level is beneficial, as described hereinabove, which iseffected by administering to a subject in need thereof a therapeuticallyeffective amount of an NO-donating compound as described hereinabove.

According to still further features in the described preferredembodiments administering of the NO-donating compound(s) is effectedorally, rectally, intravenously, topically, intranasally, intradermally,transdermally, subcutaneously, intramuscularly, intrperitoneally,intraperitoneally, by inhalation or by intrathecal catheter.

According to still further features in the described preferredembodiments the NO-donating compound is administered either per se or asa part of the pharmaceutical composition described hereinabove.

According to still further features in the described preferredembodiments the therapeutically effective amount of the NO-donatingcompound(s) ranges between about 0.01 mg/kg body and about 5 mg/kg body.

According to still further features in the described preferredembodiments the method according to this aspect of the present inventionfurther comprises administering to the subject an additional activeingredient, which is capable of treating or preventing the medicalcondition described hereinabove.

According to still another aspect of the present invention there isprovided a method of synthesizing a compound having the general FormulaI presented hereinabove. The method comprising:

providing a thioamide having a general Formula II:

wherein the thioamide is preferably selected from the group consistingof N,N-dimethylthiourea, thiobenzamide, thiourea, N-allylthiourea,acetylthiourea, N-ethylthiourea, N,N-dimethylthiourea,alpha-naphthylthiourea, 1-[(3,5-bis-trifluoromethyl)phenyl]thiourea anddithiooxalamide;

providing a reactive compound having the general Formula III:

wherein:

L is a leaving group, preferably selected from the group consisting ofhalide, alkoxy, aryloxy, amine, hydroxy, azide, nitro, cyano,thiocyanate, O-carboxylate, thiohydroxy and sulfonate, and morepreferably is halide;

Z and B are as defined above; and

W is a pre-nitratable group, preferably selected from the groupconsisting of alkoxy, aryloxy, thioalkoxy, thioaryloxy, silanoxy,silicate and O-carboxylate;

reacting the thioamide having the general Formula II and the compoundhaving the general Formula III, to thereby generate a thiazolederivative having a general Formula IV:

wherein:

A, X, B and Z are as defined above; and

U is a nitratable group, preferably selected from the group consistingof hydroxy and thiohydroxy; and

converting the nitratable group into an NO-releasing group.

Preferably, converting the nitratable group into an NO-releasing groupis effected by reacting the thiazole derivative of Formula IV with anitrating agent, which contains the NO-releasing moiety. Furtherpreferably, the NO-releasing moiety is ONO₂ and the nitrating agent isnitric acid.

In a preferred embodiment of this aspect of the present invention, thecompound of Formula III is 5-acetoxy-3-chloro-2-pentanone.

According to still further features in the described preferredembodiments providing the thioamide comprises:

providing an amide having a general Formula V:

wherein:

X and A are as defined above;

whereby the amide is preferably selected from the group consisting ofpropionamide, acetamide, isobutyramide, L-(−)-lactamide,trifluoroacetamide, carbamic acid methyl ester, hexanedioic aciddiamide, piperidine-4-carboxylic acid amide, thionicotinamide,naproxenamide, 4-(trifluoromethyl)-thiobenzamide, azodicarbonamide,2-(4-isobutyl-phenyl)-propionamide, isonicotinamide,2,2,2-trifluoroacetamide, glycinamide, 4-aminobenzamide,2,3,4,5,6-pentafluorobenzamide, 2-aminobenzamide, ethyloxamate,2,6-difluorobenzamide, N-phenylurea, 2,4-dichlorophenylacetamide,2,4-dichlorophenoxyacetmide, 2-phenylbutyamide, azodicarbonamide,3,5-difluorobenzamide, DL-lipoamide, Rubeanic acid, adpamide,aalonamide, acrylamide and 2-hydroxy-benzamide; and

reacting the amide with a thiolating agent.

Preferably, the thiolating agent is phosphorous pentasulfide.

According to still further features in the described preferredembodiments the pre-nitratable group is acetate and the nitratable groupis hydroxy.

According to an additional aspect of the present invention there isprovided a method of synthesizing a compound having the general FormulaI hereinabove, in which A is a biocleavable moiety.

The method according to this aspect of the present invention comprises:

providing a thiazole having a general Formula VI:

wherein:

Z, B and U are as defined above; and

Q is a first reactive group;

whereby the thiazole is preferably2-(2-amino-4-methyl-thiazol-5-yl)-ethanol or5-(2-hydroxy-ethyl)-4-methyl-thiazole-2-carboxylic;

providing a compound the general Formula VII:X—K  Formula VIIwherein:

X is as defined above; and

K is a second reactive group;

whereby this compound is preferably selected from consisting of2-(6-methoxy-naphthalen-2-yl)-propionic acid,4-[1,2]dithiolan-3-yl-butyric acid, 2-(4-isobutyl-phenyl)-propionicacid, nicotinic acid, 1-oxy-nicotinic acid, 2,6-difluoro-benzoic acid,phthalazin-1-yl-hydrazine, 3-chloro-propene, 4-acetylamino-benzoic acid,hexadecanoic acid, 2-acetoxy-benzoic acid, pyrrolidine-2-carboxylicacid, (2,4-dichloro-phenyl)-acetic acid, (2,4-dichloro-phenoxy)-aceticacid and17-hydroxy-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-cyclopenta[a]phenanthren-3-one;

reacting the thiazole having the general Formula VI and the compoundhaving the general Formula VII, to thereby generate a thiazolederivative having a general Formula IV:

wherein:

A, X, B and Z are as defined above; and

U is a nitratable group as described hereinabove; and

converting the nitratable group into an NO-releasing group, as describedhereinabove.

According to still further features in the described preferredembodiments, the biocleavable moiety is selected from the groupconsisting of amide, carboxylate, carbonate, carbamate, phosphate,hydrazide, thiohydrazide, disulfide, epoxide, peroxo and methyleneamine.

According to still further features in the described preferredembodiments the first reactive group and the second reactive group areeach independently selected from the group consisting of amine, halide,acyl-halide, sulfonate, sulfoxides, phosphate, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, isocyanate,sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate,O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide,N-amide, guanyl, guanidine and hydrazine. Preferably, the first reactivegroup and the second reactive group are each independently selected fromthe group consisting of amine, hydrazine, alkoxy, halide andcarboxylate.

According to still an additional aspect of the present invention thereis provided a medical device which includes an NO-donating compound asdescribed hereinabove and a delivery system configured for deliveringthe NO-donating compound to a bodily site of a subject.

According to still further features in the described preferredembodiments the bodily site in selected from the group consisting ofskin, scalp, a dermal layer, an eye, an ear, a small intestines tissue,a large intestines tissue, a kidney, a pancreas, a liver, a digestivetract, a respiratory tract, a bone, a bone marrow tissue, a mucosalmembrane, a nasal membrane, the blood system, a blood vessel, a muscle,a pulmonary cavity, an artery, a vein, a capillary, a heart, a heartcavity, a male reproductive organ, a female reproductive organ and avisceral organ.

According to still further features in the described preferredembodiments the NO-donating compound forms a part of the pharmaceuticalcomposition described hereinabove.

According to still further features in the described preferredembodiments the delivering is effected by inhalation and the deliverysystem is selected from the group consisting of a metered dose inhaler,a respirator, a nebulizer inhaler, a dry powder inhaler, an electricwarmer, a vaporizer, an atomizer and an aerosol generator.

According to still further features in the described preferredembodiments the delivering is effected transdermally and the deliverysystem is selected from the group consisting of an adhesive plaster anda skin patch.

According to still further features in the described preferredembodiments the delivering is effected topically and the delivery systemis selected from the group consisting of an adhesive strip, a bandage,an adhesive plaster, a wound dressing and a skin patch.

According to still further features in the described preferredembodiments the delivering is effected by implanting the medical devicein a bodily organ.

According to still further features in the described preferredembodiments the delivery system further includes a biocompatible matrix.Preferably the biocompatible matrix includes a biodegradable polymer.Further preferably, the biocompatible matrix includes a slow releasecarrier.

According to still further features in the described preferredembodiments the delivery system is selected from the group consisting ofan aortic aneurysm graft device, an atrioventricular shunt, a catheter,a defibrilator, a heart valve, a hemodialysis catheter, a hemodialysisgraft, an indwelling arterial catheter, an indwelling venous catheter, aneedle, a pacemaker, a pacemaker lead, a patent foramen ovale septalclosure device, a stent, a stent graft, a suture, a synthetic vasculargraft, a thread, a tube, a vascular anastomosis clip, a vascularaneurysm occluder, a vascular clip, a vascular prosthetic filter, avascular sheath and a drug delivery port, a venous valve and a wire.

According to another aspect of the present invention there is provided ause of an NO-donating compound as described herein in the treatment of amedical condition in which modulating an NO level is beneficial, asdetailed herein.

According to another aspect of the present invention there is provided ause of an NO-donating compound as described herein for the preparationof a medicament for treating or preventing a medical condition in whichmodulating an NO level is beneficial, as detailed herein.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a novel class of NO-donatingagents, which are both highly efficient in treating NO-deficiencies andare not associated with drug tolerance, and which may further havetherapeutically active agent attached thereto.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

The term “comprising” means that other steps and ingredients that do notaffect the final result can be added. This term encompasses the terms“consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the chemical, pharmacological, biological, biochemicaland medical arts.

The term “active ingredient” refers to a pharmaceutical agent includingany natural or synthetic chemical substance that subsequent to itsapplication has, at the very least, at least one desired pharmaceuticalor therapeutic effect.

The term “therapeutically effective amount” or “pharmaceuticallyeffective amount” denotes that dose of an active ingredient or acomposition comprising the active ingredient that will provide thetherapeutic effect for which the active ingredient is indicated, herein,modulating and preferably elevating an NO level.

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 presents a thin-layer chromatogram showing the retention times(obtained using ethyl acetate as eluent) and the presence of a nitrateester moiety (demonstrated by staining with diphenylamine) ofglyceryltrinitrate and exemplary NO-donor compounds according to thepresent invention, Pet-7 Pet-8, Pet-12, Pet-13;

FIG. 2 presents a plot demonstrating the vasorelaxation effect inducedby Pet-2, an exemplary NO-donor according to the present invention, inisolated rat aorta pre-treated in vitro with 1 μM epinephrine (errorbars represent the mean±standard errors, n=5-8);

FIG. 3 presents comparative plots demonstrating the superiorvasorelaxation effect induced by Pet-2, an exemplary NO-donor accordingto the present invention (squares), as compared with glyceryltrinitrate(diamonds) in isolated rat aorta pre-treated in vitro with 1 μMepinephrine (error bars represent the mean±standard errors, n=4);

FIG. 4 presents comparative plots demonstrating the superiorvasorelaxation effect induced by of Pet-3 (n=8, diamonds), an exemplaryNO-donor according to the present invention, as compared withglyceryltrinitrate (n=4, squares) in isolated rat aorta pre-treated invitro with 1 μM epinephrine (error bars represent the mean±standarderrors);

FIG. 5 presents comparative plots demonstrating the superior (10-foldhigher) vasorelaxation effect induced by of Pet-7 (n=4, diamonds), anexemplary NO-donor according to the present invention, as compared withglyceryltrinitrate (n=4, squares) in isolated rat aorta pre-treated invitro with 1 μM epinephrine (error bars represent the mean±standarderrors);

FIG. 6 presents comparative plots demonstrating the superiorvasorelaxation effect induced by of Pet-8 (diamonds), an exemplaryNO-donor according to the present invention, as compared withglyceryltrinitrate (squares) in isolated rat aorta pre-treated in vitrowith 1 μM epinephrine (error bars represent the mean±standard errors);

FIG. 7 presents comparative plots demonstrating the vasorelaxationeffect induced by of Pet-12 (Pet12-Ni, diamonds), an exemplary NO-donoraccording to the present invention, as compared with the correspondingpre-nitrated compound (Pet12, squares) in isolated rat aorta pre-treatedin vitro with 1 μM epinephrine (error bars represent the mean±standarderrors, n=6);

FIG. 8 presents comparative plots demonstrating the vasorelaxationeffect induced by Pet-24 (diamonds), an exemplary NO-donor according tothe present invention, as compared with glyceryltrinitrate (squares) inisolated rat aorta pre-treated in vitro with 1 μM epinephrine (errorbars represent the mean±standard errors, n=4);

FIG. 9 presents comparative plots demonstrating the superiorvasorelaxation effect induced by of Pet-43 (diamonds), an exemplaryNO-donor according to the present invention, as compared withglyceryltrinitrate (squares) in isolated rat aorta pre-treated in vitrowith 1 μM epinephrine (error bars represent the mean±standard errors,n=4);

FIG. 10 presents comparative plots demonstrating the superiorvasorelaxation effect induced by of Pet-59 (diamonds), an exemplaryNO-donor according to the present invention, as compared withglyceryltrinitrate (squares) in isolated rat aorta pre-treated in vitrowith 1 μM epinephrine (error bars represent the mean±standard errors);

FIG. 11 presents comparative plots demonstrating the vasorelaxationeffect induced by of Pet-147 (diamonds), an exemplary NO-donor accordingto the present invention, as compared with glyceryltrinitrate (squares)in isolated rat aorta pre-treated in vitro with 1 μM epinephrine (errorbars represent the mean±standard errors);

FIG. 12 presents comparative plots demonstrating the superiorvasorelaxation effect induced by of Pet-149 (diamonds), an exemplaryNO-donor according to the present invention, as compared withglyceryltrinitrate (squares) in isolated rat aorta pre-treated in vitrocontraction 1 μM epinephrine (error bars represent the mean±standarderrors);

FIG. 13 presents comparative plots demonstrating the superiorvasorelaxation effect induced by of Pet-152 (n=8, diamonds), anexemplary NO-donor according to the present invention, as compared withglyceryltrinitrate (n=4, squares) in isolated rat aorta pre-treated invitro with 1 μM epinephrine (error bars represent the mean±standarderrors);

FIG. 14 presents comparative plots demonstrating the vasorelaxationeffect induced by of Pet-154 (n=8, diamonds), an exemplary NO-donoraccording to the present invention, as compared with glyceryltrinitrate(n=4, squares) in isolated rat aorta pre-treated in vitro with 1 μMepinephrine (error bars represent the mean±standard errors);

FIG. 15 presents comparative plots showing the vasorelaxation effectinduced by GTN in isolated rat aorta pre-treated in vitro withepinephrine (control, squares) and in isolated rat aorta pre-treated invitro with epinephrine and then with GTN (diamonds), and demonstratingthe tolerance induced by treatment with GTN (error bars represent themean±standard errors);

FIG. 16 presents comparative plots showing the vasorelaxation effectinduced by Pet-3, an exemplary NO-donor according to the presentinvention, in isolated rat aorta pre-treated in vitro with epinephrine(control, diamonds) and in isolated rat aorta pre-treated in vitro withepinephrine and then with 0.44 mM Pet-3 (squares), and demonstrating theabsence of tolerance induction following treatment with the NO-donors ofthe present invention, (error bars represent the mean±standard errors,n=4);

FIG. 17 presents comparative plots showing the vasorelaxation effectinduced by Pet-7, an exemplary NO-donor according to the presentinvention, in isolated rat aorta pre-treated in vitro with epinephrine(control, diamonds) and in isolated rat aorta pre-treated in vitro withepinephrine and then with 0.44 mM Pet-7 (squares), and demonstrating theabsence of tolerance induction following treatment with the NO-donors ofthe present invention, (error bars represent the mean±standard errors,n=4);

FIG. 18 presents comparative plots showing the vasorelaxation effectinduced by Pet-12, an exemplary NO-donor according to the presentinvention, in isolated rat aorta pre-treated in vitro with epinephrine(control, diamonds) and in isolated rat aorta pre-treated in vitro withepinephrine and then with 0.44 mM Pet-12 (squares), and demonstratingthe absence of tolerance induction following treatment with theNO-donors of the present invention (error bars represent themean±standard errors);

FIG. 19 presents comparative plots showing the vasorelaxation effectinduced by Pet-12 (diamonds), an exemplary NO-donor according to thepresent invention, and GTN (squares) in isolated rat aorta pre-treatedin vitro with epinephrine and then with GTN, and demonstrating theabsence of tolerance induction effect of the NO-donors of the presentinvention following treatment with GTN (error bars represent themean±standard errors);

FIG. 20 presents comparative plots showing the vasorelaxation effectinduced by Pet-12 (squares), an exemplary NO-donor according to thepresent invention, and GTN (squares) in isolated rat aorta pre-treatedin vitro with epinephrine and then with Pet-12, and demonstrating theabsence of tolerance induction effect of NO-donors of the presentinvention following treatment with Pet-12 (error bars represent themean±standard errors);

FIG. 21 presents comparative plots showing the vasorelaxation effectinduced by Pet-24, an exemplary NO-donor according to the presentinvention, in isolated rat aorta pre-treated in vitro with epinephrine(control, diamonds) and in isolated rat aorta pre-treated in vitro withepinephrine and then with 0.44 mM Pet-24 (squares), and demonstratingthe absence of tolerance induction following treatment with theNO-donors of the present invention, (error bars represent themean±standard errors);

FIG. 22 presents comparative plots showing the vasorelaxation effectinduced by Pet-43, an exemplary NO-donor according to the presentinvention, in isolated rat aorta pre-treated in vitro with epinephrine(control, diamonds) and in isolated rat aorta pre-treated in vitro withepinephrine and then with 0.44 mM Pet-43 (squares), and demonstratingthe absence of tolerance induction following treatment with theNO-donors of the present invention, (error bars represent themean±standard errors);

FIG. 23 presents comparative plots showing the vasorelaxation effectinduced by Pet-149, an exemplary NO-donor according to the presentinvention, in isolated rat aorta pre-treated in vitro with epinephrine(control, diamonds) and in isolated rat aorta pre-treated in vitro withepinephrine and then with 0.44 mM Pet-149 (squares), and demonstratingthe absence of tolerance induction following treatment with theNO-donors of the present invention, (error bars represent themean±standard errors);

FIG. 24 presents a bar graph showing the effect of GTN and Pet-2, anexemplary NO-donor according to the present invention, on theaccumulation of cyclic-GMP (determined by radioimmunoassay) in isolatedaorta of rats pre-treated in vivo with GTN and Pet-2 respectively,demonstrating the tolerance inducing effect of GTN and the absence oftolerance induction following administration of the NO-donors of thepresent invention (error bars represent the mean±standard errors, n=8);

FIG. 25 presents a bar graph showing the effect of GTN, Pet-3, Pet-7 andPet-8, exemplary NO-donors according to the present invention, on theaccumulation of cyclic-GMP (determined by radioimmunoassay) in isolatedrat aorta, demonstrating the vasorelaxation effect of the NO-donors ofthe present invention (error bars represent the mean±standard errors,n=8);

FIG. 26 presents a bar graph showing the effect of Pet-12, an exemplaryNO-donor according to the present invention, on the accumulation ofcyclic-GMP (determined by radioimmunoassay) in isolated aorta of ratstreated in vivo with Pet-12, demonstrating the absence of toleranceinduction following administration of the NO-donors of the presentinvention (error bars represent the mean±standard errors, n=8);

FIG. 27 presents a bar graph showing the effect of GTN, Pet-24 andPet-149, exemplary NO-donors according to the present invention, on theaccumulation of cyclic-GMP (determined by radioimmunoassay) in isolatedrat aorta, demonstrating the vasorelaxation effect of the NO-donors ofthe present invention (error bars represent the mean±standard errors,n=8);

FIG. 28 presents a graph demonstrating the vasorelaxation effect inducedby 100 micrograms Pet-7, an exemplary NO-donor according to the presentinvention, on the heightened mean arterial blood pressure of ratspre-treated in vivo with 50 micrograms phenylephrine;

FIG. 29 presents a graph demonstrating the vasorelaxation effect inducedby 50 micrograms Pet-12, an exemplary NO-donor according to the presentinvention, on the heightened mean arterial blood pressure of ratspre-treated in vivo with 50 micrograms phenylephrine;

FIG. 30 presents a graph demonstrating the vasorelaxation effect inducedby 10 micrograms and thereafter by 50 micrograms Pet-12, an exemplaryNO-donor according to the present invention, on the heightened meanarterial blood pressure of rats pre-treated in vivo with 10 microgramsphenylephrine;

FIG. 31 presents a graph demonstrating the vasorelaxation effect inducedby 10 micrograms and thereafter by 50 micrograms Pet-13, an exemplaryNO-donor according to the present invention, on the heightened meanarterial blood pressure of rats pre-treated in vivo with 10 microgramsphenylephrine;

FIG. 32 presents comparative plots demonstrating the superiorvasorelaxation effect induced by of Pet-155 (n=8, diamonds), anexemplary NO-donor according to the present invention, as compared withglyceryltrinitrate (n=4, squares) in isolated rat aorta pre-treated invitro with 1 μM epinephrine (error bars represent the mean±standarderrors);

FIG. 33 presents a bar graph showing the effect of GTN, Pet-10, Pet-147,Pet-Pet-152, Pet-154 and Pet-155, exemplary NO-donors according to thepresent invention, on the accumulation of cyclic-GMP (determined byradioimmunoassay) in isolated rat aorta, demonstrating thevasorelaxation effect of the NO-donors of the present invention (errorbars represent the mean±standard errors, n=8);

FIG. 34 presents a schematic illustration of a metered dose inhalator,an exemplary medical device according to the present invention;

FIG. 35 presents a schematic illustration of a nebulizer, an exemplarymedical device according to the present invention;

FIG. 36 presents a schematic illustration of a skin patch, an exemplarymedical device according to the present invention; and

FIG. 37 presents a schematic illustration of a stent, an exemplarymedical device according to the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a novel family of NO-donating compounds(NO-donors), which comprise one or more NO-releasing group(s) covalentlyattached to a chemical moiety, and which are designed such that when NOis released from the compound, a residue which is a naturally occurringmetabolite is formed. The novel NO-donors of the present invention arehighly efficacious and non-tolerance inducing and can therefore bebeneficially used in the treatment of a variety of medical conditionssuch as, but not limited to, ischemic heart disease, heart failure,hypertension and other cardiovascular diseases and serve asbioregulators in physiological processes such as neurotransmission,blood clotting, blood pressure, and the destruction of cancerous tumorcells. The novel NO-donors of the present invention may further includea bioactive moiety (e.g., a drug) and thus may exert a dual therapeuticactivity. The present invention is further of methods of preparing thenovel NO-donors, pharmaceutical compositions including the NO-donors,medical devices designed for various modes of delivery the NO-donors andmethods of using the NO-donors in the treatment of a variety of medicalconditions.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

As is discussed hereinabove, organic nitrate and nitrite estersrepresent a time-honored class of NO-donating agents used incardiovascular therapeutics since the nineteenth century. These agentshave direct vasoactive effects and have therefore been used to treatischemic heart disease, heart failure, and hypertension for many years.Growing evidence suggests that the cytochrome P-450 system, inconjunction with NADPH and glutathione-S-transferase activities, isrequired for the linked metabolic processes of denitration and reductionof organic nitrate esters to authentic NO [Kurz (1993) and McGuire(1998)].

However, as is further discussed hereinabove, treatment methodsutilizing these compounds are severely limited by their therapeutichalf-life, systemic absorption that is oftentimes accompanied by adversehemodynamic effects, and, above all, critical drug tolerance. Thisphenomenon of tolerance (the loss of therapeutic efficacy uponrepetitive administration) is by far the most predominant drawback ofnitrate therapy, which limits their medicinal application.

In view of these limitations, substantial efforts have been made todevelop and study novel NO-Donating agents. Unfortunately, most of theseagents were characterized as tolerance-inducing following prolongedadministration.

While conceiving the present invention, it was envisioned thatovercoming the limitations associated with the presently known NO-donorscould be achieved by the design and preparation of a novel class ofNO-donors in which upon releasing a bioactive NO a residue of anaturally occurring metabolite is formed. It was further envisioned thatsuch NO donors, when entering a biological system, would be subjected toenzymatic reactions that would result in the release of a bioactive NOand the formation of a residue of a metabolite, whereby this residue, bybeing derived from a naturally occurring metabolite, would becharacterized by inherent biocompatibility, non-toxicity, and efficientabsorption, distribution, excretion, metabolism and otherbiocompatibility related advantages. Above all, it was envisioned thatthe cleavage of such compounds into a residue that is characterized bysuch an inherent biocompatibility would prevent or decrease thedevelopment of tolerance to these compounds.

While further conceiving the present invention, it was envisioned thatby conjugating a bioactive compound to the thiazole-derived NO-donors,combined and possibly synergistic therapeutic effects could be achieved,resulting from the dual therapeutic effect of the bioactive agent andthe NO-releasing group.

While reducing the present invention to practice, a plurality ofNO-donating compounds was designed according to the underlyingprinciples outlined above and were readily synthesized. As isdemonstrated in the Examples section that follows, these compounds werefound highly efficacious in inducing vasorelaxation and reducinghypotension and were further found to be non-tolerance inducing in bothin vitro and in vivo assays.

As used herein, the phrase “non-tolerance inducing compound(s)” is meantto describe compounds which upon repetitive administration thereof donot induce tolerance thereto.

The present invention therefore provides a novel class of NO-donatingcompounds, which are also referred to herein interchangeably asNO-donors. Each of the compounds of the present invention comprises anNO-releasing group, as is detailed hereinunder and a chemical moietybeing covalently attached to the NO-releasing group.

As used herein, the phrase “chemical moiety” describes a residue, asthis term is defined hereinbelow, of an organic substance.

The chemical moiety and the NO-releasing group are selected and attachedone to the other such that upon release of NO from the compound, aresidue of a naturally occurring metabolite is formed. As is discussedhereinabove, due to the formation of such a residue, the development oftolerance to the compounds of the present invention upon repetitiveadministration thereof is prevented or at least substantially decreasedand hence the NO-donors of the present invention are highly advantageousand superior to the presently known NO-donors.

As used herein, the term “metabolite” describes a substance that istypically associated with one or more metabolic processes, that is, asubstance produced by a metabolic process, required for a metabolicprocess and/or participating in a metabolic process.

As is further detailed hereinbelow, the NO-donating compounds accordingto the present invention optionally and preferably further comprise abioactive agent residue. The bioactive residue is preferably attached,either directly or indirectly, preferably via a biocleavable moiety, tothe chemical moiety in the compound.

While further conceiving the present invention, it was envisioned thatexemplary NO-donating compounds as described above, which are designedto form a residue of vitamin B (thiamine) upon releasing NO, could bereadily tailored and synthesized.

Vitamin B₁, a water soluble vitamin having the chemical name3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium,is also known as thiamin, thiamine and aneurin. Thiamine is required byevery cell of the body to process carbohydrates, fat, and protein and toform the fuel compound adenosine triphosphate (ATP).

Thiamin consists of a pyrimidine ring and a thiazole ring connected by asingle-carbon bridging moiety, whereby the nitrogen in the thiazole ringbeing positively charged. It serves as a coenzyme for thedecarboxylation of pyruvate and the oxidation of alpha keto-glutamicacid.

The enzyme thiamin pyrophosphatekinase and adenosine triphosphate (ATP)convert thiamin into its metabolically active coenzyme form, thiaminpyrophosphate (TPP), which is also referred to in the art as thiaminediphosphate (TDP) and cocarboxylase. The reaction center of TPP is therelatively acidic proton on carbon 2 of the thiazole ring, which has thecapacity to form a carbanion, whereby the latter readily undergoesnucleophilic addition to carbonyl groups. In the form of TPP, thiaminfunctions in the oxidative decarboxylation of alpha-keto acids, such aspyruvate and alpha-ketoglutarate, as a coenzyme for alpha-ketoaciddehydrogenases. In addition TPP functions in the transketolase reactionof the pentose phosphate pathway as a coenzyme for transketolases. Bothtypes of enzymes, alpha-ketoacid dehydrogenases and transketolases,cleave a carbon-carbon bond adjacent to a carbonyl group, releasingeither carbon dioxide or an aldehyde. In the case of alpha-ketoaciddehydrogenases, the decarboxylation product is transferred to coenzyme A(CoA). Transketolases cleaves the carbon-carbon bond adjacent to thecarbonyl group of an alpha-ketosugar to give an activated glycoaldehyde.The glycoaldehyde is then combined with an aldose molecule to yield anew ketose. All known TPP dependent enzymes also require a divalentcation, commonly Mg²⁺.

Thiamine thus plays an important role in glucose metabolism and furtherappears to be involved in nerve transmission and/or excitation.

As thiamine is involved in numerous biological pathways, it was assumedthat any residue thereof would be characterized by the inherentbiocompatibility described above. It was further assumed that themetabolic pathways of vitamin B₁ described hereinabove could participatein releasing a bioactive NO from an NO-releasing group that is attachedto a thiamine residue, similarly to the release of a phosphate group ofTPP.

In view of the above, a representative model of NO-donating compoundsaccording to the present invention, in which each of the compounds ofthe present invention comprises an NO-releasing group that is covalentlyattached to a thiamine-derived thiazole ring, has been designed.

Derivatives of thiazole are well-known in the art and are readilysynthesized by well-established procedures. As is exemplified in theExamples section that follows, by selecting a suitable synthesis of athiamine-derived thiazole, a variety of chemical parameters can beeasily tailored, thus enabling the design and preparation of versatilethiazole-derived NO-donating compounds.

Hence, according to a preferred embodiment of the present invention,each of the NO-donating compounds described herein is designed such thatupon release of NO, a residue of thiamine is formed. Each of theNO-donating compounds according to this embodiment of the presentinvention therefore include an NO-releasing group, as is detailedhereinunder, being covalently attached to a thiamine-derived thiazolering.

Thiazole derivatives that include an NO-releasing group have only beenreported hitherto in U.S. Pat. No. 6,677,374, which teaches nitrateester-containing compounds for use as neuroprotective therapeuticagents. Two of the compounds disclosed in this patent include a thiazolering: 1-(4-methylthiazol-5-yl)ethane-1,2-diyl dinitrate and2-(4-methylthiazol-5-yl)ethyl nitrate. These compounds were prepared bynitrating two commercially available, arbitrarily selected, thiazoles,5-(1,2-dihydroxyethyl)-4-methylthiazole and4-methyl-5-(2-hydroxyethyl)thiazole. Nevertheless, this patent, by notbeing directed to NO-donating thiazole derivatives, fails to teach thedesign and preparation of various modifications of such derivatives, andparticularly fails to address the beneficial effects of such derivativesin various applications and particularly the design and use of suchthiazole derivatives as NO-donating compounds which prevent or decreasetolerance, due to the formation of a residue of a thiamine metabolite.

The NO-donating compounds according to this embodiment of the presentinvention include, for example, a thiamine nitrated derivative, in whichthe hydroxyl end group at position 5 has been replaced by a —ONO₂ group(see, for example, Pet-68 in Tables 1 and 2 below), and/or apharmaceutically acceptable salt thereof.

Alternatively, the NO-donating compounds according to this embodiment ofthe present invention, include a thiamine analog, which has athiamine-derived thiazole ring as its basic structural unit and/or apharmaceutically acceptable salt thereof. Such NO-donating compoundsaccording to the present invention are referred to hereininterchangeably as thiazole-derived or thiazole-based compounds and arecollectively represented by the general formula I:

wherein:

A is selected from the group consisting of alkenyl, alkoxy, alkyl,alkynyl, amine, amine-oxide, aryl, aryloxy, azo, borate, C-amide,carbonyl, C-carboxylate, C-thiocarboxylate, cycloalkyl, diazo,disulfide, guanidine, guanyl, haloalkyl, heteroalicyclic, heteroaryl,hydrazine, N-amide, N-carbamate, N-dithiocarbamate, nitro,N-sulfonamide, N-thiocarbamate, O-carbamate, O-carboxylate,O-thiocarbamate, O-thiocarboxylate, oxime, oxygen, peroxo, phosphate,phosphine-oxide, phosphine-sulfide, phosphinyl, phosphite, phosphonate,pyrophosphate, S-dithiocarbamate, silaza, silicate, siloxy, silyl,S-sulfonamide, sulfate, sulfite, sulfonate, sulfoxide, sulfur,thioalkoxy, thioaryloxy, thiocarbonyl, thiophosphate, thiosulfate,thiosulfite, thiourea, triphosphate, urea, a biocleavable moiety and anycombination thereof, or absent;

X is selected from the group consisting of acyl halide, alkenyl, alkoxy,alkyl, alkynyl, amine, amine-oxide, aryl, aryloxy, azo, borate, C-amide,carbonyl, C-carboxylate, C-thiocarboxylate, cyano, cycloalkyl, diazo,disulfide, guanidine, guanyl, halide, haloalkyl, heteroalicyclic,heteroaryl, hydrazine, hydrogen, hydroxy, N-amide, N-carbamate,N-dithiocarbamate, nitro, N-sulfonamide, N-thiocarbamate, O-carbamate,O-carboxylate, O-thiocarbamate, O-thiocarboxylate, oxime, peroxo,phosphate, phosphine-oxide, phosphine-sulfide, phosphinyl, phosphite,phosphonate, pyrophosphate, S-dithiocarbamate, silaza, silicate, siloxy,silyl, S-sulfonamide, sulfate, sulfite, sulfonate, sulfoxide,thioalkoxy, thioaryloxy, thiocarbonyl, thiohydroxy, thiophosphate,thiosulfate, thiosulfite, thiourea, triphosphate, urea, a bioactiveagent residue, a moiety containing at least one NO-releasing group, asubstituted or unsubstituted thiazole and any combination thereof;

B is selected from the group consisting of a saturated or unsaturated,substituted or unsubstituted alkylene chain having 1-20 carbon atoms,and a saturated or unsaturated, substituted or unsubstituted alkylenechain having 1-20 carbon atoms interrupted by at least one heteroatom,whereby the at least one heteroatom comprises oxygen, sulfur, nitrogen,phosphor, silicon and any combination thereof;

Y is an NO-releasing group; and

Z is selected from the group consisting of hydrogen, alkyl, alkenyl,alkynyl, amine, cycloalkyl, heteroalicyclic, aryl, heteroaryl, halide,haloalkyl, hydroxy, thiohydroxy, alkoxy, thioalkoxy, aryloxy andthioaryloxy.

It will be appreciated by one of skills in the art that the feasibilityof each of the variables (denoted as A, B, X, Y and Z) to be located atthe indicated positions depends on the valency and chemicalcompatibility of the substituent, the substituted position and othersubstituents. Hence, the present invention is aimed at encompassing allthe feasible substituents for any position.

As used herein, the term “amine” describes both a —NR′R″ group and a—NR′— group, wherein R′ and R″ are each independently hydrogen, alkyl,cycloalkyl, aryl, as these terms are defined hereinbelow.

The amine group can therefore be a primary amine, where both R′ and R″are hydrogen, a secondary amine, where R′ is hydrogen and R″ is alkyl,cycloalkyl or aryl, or a tertiary amine, where each of R′ and R″ isindependently alkyl, cycloalkyl or aryl.

Alternatively, R′ and R″ can each independently be hydroxyalkyl,trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate,hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano,nitro, azo, sulfonamide, carbonyl, C-carboxylate, O-carboxylate,N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate,O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine.

The term “amine” is used herein to describe a —NR′R″ group in caseswhere the amine is an end group, as defined hereinunder, and is usedherein to describe a —NR′— group in cases where the amine is a linkinggroup.

Herein throughout, the phrase “end group” describes a group (asubstituent) that is attached to another moiety in the compound via oneatom thereof.

The phrase “linking group” describes a group (a substituent) that isattached to another moiety in the compound via two or more atomsthereof.

The term “alkyl” describes a saturated aliphatic hydrocarbon includingstraight chain and branched chain groups. Preferably, the alkyl grouphas 1 to 20 carbon atoms. Whenever a numerical range; e.g., “1-20”, isstated herein, it implies that the group, in this case the alkyl group,may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up toand including 20 carbon atoms. More preferably, the alkyl is a mediumsize alkyl having 1 to 10 carbon atoms. Most preferably, unlessotherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbonatoms. The alkyl group may be substituted or unsubstituted. Substitutedalkyl may have one or more substituents, whereby each substituent groupcan independently be, for example, hydroxyalkyl, trihaloalkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine,halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide,C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea,thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidineand hydrazine.

The alkyl group can be an end group, as this phrase is definedhereinabove, wherein it is attached to a single adjacent atom, or alinking group, as this phrase is defined hereinabove, which connects twoor more moieties via at least two carbons in its chain.

The term “cycloalkyl” describes an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereone or more of the rings does not have a completely conjugatedpi-electron system. The cycloalkyl group may be substituted orunsubstituted. Substituted cycloalkyl may have one or more substituents,whereby each substituent group can independently be, for example,hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl,heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide,phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate,O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea,N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine andhydrazine. The cycloalkyl group can be an end group, as this phrase isdefined hereinabove, wherein it is attached to a single adjacent atom,or a linking group, as this phrase is defined hereinabove, connectingtwo or more moieties at two or more positions thereof.

The term “aryl” describes an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. The aryl groupmay be substituted or unsubstituted. Substituted aryl may have one ormore substituents, whereby each substituent group can independently be,for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl,aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide,phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate,O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea,N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine andhydrazine. The aryl group can be an end group, as this term is definedhereinabove, wherein it is attached to a single adjacent atom, or alinking group, as this term is defined hereinabove, connecting two ormore moieties at two or more positions thereof.

The term “amine-oxide” describes a —N(OR′)(R″) or a —N(OR′)— group,where R′ and R″ are as defined herein. This term refers to a —N(OR′)(R″)group in cases where the amine-oxide is an end group, as this phrase isdefined hereinabove, and to a —N(OR′)— group in cases where theamine-oxime is an end group, as this phrase is defined hereinabove.

The term “halide” and “halo” describes fluorine, chlorine, bromine oriodine.

The term “haloalkyl” describes an alkyl group as defined above, furthersubstituted by one or more halide.

The term “sulfate” describes a —O—S(═O)₂—OR′ end group, as this term isdefined hereinabove, or an —O—S(═O)₂—O— linking group, as these phrasesare defined hereinabove, where R′ is as defined hereinabove.

The term “thiosulfate” describes a —O—S(═S)(═O)—OR′ end group or a—O—S(═S)(═O)—O— linking group, as these phrases are defined hereinabove,where R′ is as defined hereinabove.

The term “sulfite” describes an —O—S(═O)—R′ end group or a —O—S(═O)—O—group linking group, as these phrases are defined hereinabove, where R′is as defined hereinabove.

The term “thiosulfite” describes a —S(═S)—O—R′ end group or an —S(═S)—Ogroup linking group, as these phrases are defined hereinabove, where R′is as defined hereinabove.

The term “sulfinate” describes a —S(═O)—OR′ end group or an —S(═O)—group linking group, as these phrases are defined hereinabove, where R′is as defined hereinabove.

The term “sulfoxide” or “sulfinyl” describes a —S(═O)R′ end group or an—S(═O)— linking group, as these phrases are defined hereinabove, whereR′ is as defined hereinabove.

The term “sulfonate” describes a —S(═O)₂—R′ end group or an —S(═O)₂—linking group, as these phrases are defined hereinabove, where R′ is asdefined herein.

The term “S-sulfonamide” describes a —S(═O)₂—NR′R″ end group or a—S(═O)₂—NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “N-sulfonamide” describes an R′S(═O)₂—NR″— end group or a—S(═O)₂—NR′— linking group, as these phrases are defined hereinabove,where R′ and R″ are as defined herein.

The term “disulfide” refers to a —S—SR′ end group or a —S—S— linkinggroup, as these phrases are defined hereinabove, where R′ is as definedherein.

The term “phosphonate” describes a —P(═O)(OR′)(OR″) end group or a—P(═O)(OR′)(O)— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “thiophosphonate” describes a —P(═S)(OR′)(OR″) end group or a—P(═S)(OR′)(O)— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “phosphinyl” describes a —PR′R″ end group or a —PR′— linkinggroup, as these phrases are defined hereinabove, with R′ and R″ asdefined hereinabove.

The term “phosphine oxide” describes a —P(═O)(R′)(R″) end group or a—P(═O)(R′)— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “phosphine sulfide” describes a —P(═S)(R′)(R″) end group or a—P(═S)(R′)— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “phosphite” describes an —O—PR′(═O)(OR″) end group or an—O—PH(═O)(O)— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “carbonyl” or “carbonate” as used herein, describes a —C(═O)—R′end group or a —C(═O)— linking group, as these phrases are definedhereinabove, with R′ as defined herein.

The term “thiocarbonyl” as used herein, describes a —C(═S)—R′ end groupor a —C(═S)— linking group, as these phrases are defined hereinabove,with R′ as defined herein.

The term “oxime” describes a ═N—OH end group or a ═N—O— linking group,as these phrases are defined hereinabove.

The term “hydroxyl” describes a —OH group.

The term “alkoxy” describes both an —O-alkyl and an —O-cycloalkyl group,as defined herein.

The term “aryloxy” describes both an —O-aryl and an —O-heteroaryl group,as defined herein.

The term “thiohydroxy” describes a —SH group.

The term “thioalkoxy” describes both a —S-alkyl group, and a—S-cycloalkyl group, as defined herein.

The term “thioaryloxy” describes both a —S-aryl and a —S-heteroarylgroup, as defined herein.

The term “cyano” describes a —C≡N group.

The term “isocyanate” describes an —N═C═O group.

The term “nitro” describes an —NO₂ group.

The term “acyl halide” describes a —(C═O)R″″ group wherein R″″ ishalide, as defined hereinabove.

The term “azo” or “diazo” describes an —N═NR′ end group or an —N═N—linking group, as these phrases are defined hereinabove, with R′ asdefined hereinabove.

The term “peroxo” describes an —O—OR′ end group or an —O—O— linkinggroup, as these phrases are defined hereinabove, with R′ as definedhereinabove.

The term “C-carboxylate” describes a —C(═O)—OR′ end group or a —C(═O)—O—linking group, as these phrases are defined hereinabove, where R′ is asdefined herein.

The term “O-carboxylate” describes a —OC(═O)R′ end group or a —OC(═O)—linking group, as these phrases are defined hereinabove, where R′ is asdefined herein.

The term “C-thiocarboxylate” describes a —C(═S)—OR′ end group or a—C(═S)—O— linking group, as these phrases are defined hereinabove, whereR′ is as defined herein.

The term “O-thiocarboxylate” describes a —OC(═S)R′ end group or a—OC(═S)— linking group, as these phrases are defined hereinabove, whereR′ is as defined herein.

The term “N-carbamate” describes an R″OC(═O)—NR′— end group or a—OC(═O)—NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “O-carbamate” describes an —OC(═O)—NR′R″ end group or an—OC(═O)—NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “O-thiocarbamate” describes a —OC(═S)—NR′R″ end group or a—OC(═S)—NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “N-thiocarbamate” describes an R″OC(═S)NR′— end group or a—OC(═S)NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “S-dithiocarbamate” describes a —SC(═S)—NR′R″ end group or a—SC(═S)NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “N-dithiocarbamate” describes an R″SC(═S)NR′— end group or a—SC(═S)NR′— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “urea”, which is also referred to herein as “ureido”, describesa —NR′C(═O)—NR″R′″ end group or a —NR′C(═O)—NR″— linking group, as thesephrases are defined hereinabove, where R′ and R″ are as defined hereinand R′″ is as defined herein for R′ and R″.

The term “thiourea”, which is also referred to herein as “thioureido”,describes a —NR′—C(═S)—NR″R′″ end group or a —NR′—C(═S)—NR″— linkinggroup, with R′, R″ and R′″ as defined herein.

The term “C-amide” describes a —C(═O)—NR′R″ end group or a —C(═O)—NR′—linking group, as these phrases are defined hereinabove, where R′ and R″are as defined herein.

The term “N-amide” describes a R′C(═O)—NR″— end group or a R′C(═O)—N—linking group, as these phrases are defined hereinabove, where R′ and R″are as defined herein.

The term “guanyl” describes a R′R″NC(═N)— end group or a —R′NC(═N)—linking group, as these phrases are defined hereinabove, where R′ and R″are as defined herein.

The term “guanidine” describes a —R′NC(═N)—NR″R′ end group or a—R′NC(═N)—NR″— linking group, as these phrases are defined hereinabove,where R′, R″ and R′″ are as defined herein.

The term “hydrazine” describes a —NR′—NR″R′″ end group or a —NR′—NR″—linking group, as these phrases are defined hereinabove, with R′, R″,and R′″ as defined herein.

The term “silyl” describes a —SiR′R″R′″ end group or a —SiR′R″— linkinggroup, as these phrases are defined hereinabove, whereby each of R′, R″and R′″ are as defined herein.

The term “siloxy” describes a —Si(OR′)R″R′″ end group or a —Si(OR′)R″—linking group, as these phrases are defined hereinabove, whereby each ofR′, R″ and R′″ are as defined herein.

The term “silaza” describes a —Si(NR′R″)R′″ end group or a —Si(NR′R″)—linking group, as these phrases are defined hereinabove, whereby each ofR′, R″ and R′″ is as defined herein.

The term “silicate” describes a —O—Si(OR′)(OR″)(OR′″) end group or a—O—Si(OR′)(OR″)— linking group, as these phrases are definedhereinabove, with R′, R″ and R′″ as defined herein.

The term “boryl” describes a —BR′R″ end group or a —BR′— linking group,as these phrases are defined hereinabove, with R′ and R″ are as definedherein.

The term “borate” describes a —O—B(OR′)(OR″) end group or a—O—B(OR′)(O—) linking group, as these phrases are defined hereinabove,with R′ and R″ are as defined herein.

The term “heteroaryl” describes a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine. The heteroaryl group may besubstituted or unsubstituted. Substituted heteroaryl may have one ormore substituents, whereby each substituent group can independently be,for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl,aryl, heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide,phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate,O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea,O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine andhydrazine. The heteroaryl group can be an end group, as this phrase isdefined hereinabove, where it is attached to a single adjacent atom, ora linking group, as this phrase is defined hereinabove, connecting twoor more moieties at two or more positions thereof. Representativeexamples are pyridine, pyrrole, oxazole, indole, purine and the like.

The term “heteroalicyclic” describes a monocyclic or fused ring grouphaving in the ring(s) one or more atoms such as nitrogen, oxygen andsulfur. The rings may also have one or more double bonds. However, therings do not have a completely conjugated pi-electron system. Theheteroalicyclic may be substituted or unsubstituted. Substitutedheteroalicyclic may have one or more substituents, whereby eachsubstituent group can independently be, for example, hydroxyalkyl,trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate,hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano,nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate,O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide,N-amide, guanyl, guanidine and hydrazine. The heteroalicyclic group canbe an end group, as this phrase is defined hereinabove, where it isattached to a single adjacent atom, or a linking group, as this phraseis defined hereinabove, connecting two or more moieties at two or morepositions thereof. Representative examples are piperidine, piperazine,tetrahydrofurane, tetrahydropyrane, morpholino and the like.

As used herein, the phrase “NO-releasing group” describes a chemicalmoiety, which is capable of generating NO either spontaneously or bymeans of chemical or enzymatic reactions. Representative examples ofsuitable NO-releasing groups according to the present invention include,without limitation, nitrate esters such as, for example, —ONO₂,S-nitrosothiol such as, for example, —SNO, diazeniumdiolates, also knownas “NONOates” such as, for example, —N(NONO⁻)— and —N(NONOH)—, andmesoionic oxatriazoles such as for example,5-amino-[1,2,3,4]oxatriazol-2-ium and,2,3,4-oxatriazolium-5-amino-3-(3,4-dichlorophenyl)-chloride. Preferably,the NO-releasing group in the compounds of the present invention,denoted as Y in the general Formula I above, is —ONO₂.

Thus, each of the compounds according to this embodiment of the presentinvention has a thiazole ring, to which an NO-releasing group isattached, preferably at position 5 of the ring. The NO-releasing groupcan be attached directly to the thiazole ring, or, preferably via aspacer.

The spacer, denoted as B in the general Formula I above, can be asaturated or unsaturated, substituted or unsubstituted hydrocarbonchain, and may optionally be interrupted by one or more heteroatom(s)such as oxygen, sulfur, nitrogen, phosphor, silicon and any combinationthereof. When the heteroatom is nitrogen, phosphor or silicon, theheteroatom is preferably substituted by e.g., hydrogen, alkyl, halide,cycloalkyl or aryl, as these terms are defined hereinabove.

The chemical structure and length of the spacer may affect thebiocompatibility, bioavailability, target specificity, and NO-releasingsensitivity of the compound.

According to preferred embodiments of this embodiment of the presentinvention, B is a non-substituted, saturated alkylene chain. Thus, B ispreferably a non-substituted alkylene chain and, more preferably, ashort alkylene chain such as, for example, methylene, ethylene andpropylene. Since in the thiazole ring of Thiamine position 5 issubstituted by a hydroxyethlene, more preferably, B is ethylene.

Alternatively, B can be a non-substituted, saturated alkylene chaininterrupted by one heteroatom and can therefore be, for example,—CH₂—CH₂—O—CH₂— (methoxy ethylene), —CH₂—CH₂—NH—CH₂—(ethyl-methyl-amine) and —CH₂—CH₂—S—CH₂— (ethyl-methylsulfanyl).

The thiazole ring may be further substituted at position 4, by variablesubstituents, denoted as Z in the general Formula I above, which mayalso be selected so as to affect the compound's pharmacokineticproperties such as biocompatibility, bioavailability, solubility andtarget specificity.

Since in the thiazole ring of thiamine position 4 is substituted by amethyl, preferably Z is an alkyl, more preferably a lower alkyl such asmethyl, ethyl and propyl, and more preferably, Z is methyl.

Each of the compounds according to this embodiment of the presentinvention further includes a moiety that is covalently attached toposition 2 thereof. This moiety, denoted as X in the general Formula Iabove, can be a chemical moiety such as, for example, acyl-halide,alkenyl, alkoxy, alkyl, alkynyl, amine, amine-oxide, aryl, aryloxy, azo,borate, C-amide, carbonyl, C-carboxylate, C-thiocarboxylate, cyano,cycloalkyl, diazo, disulfide, guanidine, guanyl, halide, haloalkyl,heteroalicyclic, heteroaryl, hydrazine, hydrogen, hydroxy, N-amide,N-carbamate, N-dithiocarbamate, nitro, N-sulfonamide, N-thiocarbamate,O-carbamate, O-carboxylate, O-thiocarbamate, O-thiocarboxylate, oxime,peroxo, phosphate, phosphine-oxide, phosphine-sulfide, phosphinyl,phosphite, phosphonate, pyrophosphate, S-dithiocarbamate, silaza,silicate, siloxy, silyl, S-sulfonamide, sulfate, sulfite, sulfonate,sulfoxide, thioalkoxy, thioaryloxy, thiocarbonyl, thiohydroxy,thiophosphate, thiosulfate, thiosulfite, thiourea, triphosphate, ureaand any combination thereof. The attachment of such moieties may furtheraffect the pharmacokinetic profile of the compound, as describedhereinabove.

Optionally, X can be a moiety containing one or more NO-releasinggroup(s). Compounds in which X contains one or more NO-releasing group,in addition to the NO-releasing group in B (see, Formula I above), mayexert enhanced capacity to elevate bioactive NO levels. Furthermore, thepresence of more than one NO-releasing group in the same compoundenables to incorporate therein different NO-releasing groups, which maybe susceptible to more than one NO-releasing bio- and/or chemo-mechanismand thus further enhance the capacity of the compound to elevate NOlevels.

As is detailed in the Examples section that follows, representativeexamples of compounds in which X is a moiety containing an NO-releasinggroup have been successfully prepared. These include1,4-bis-[4-methyl-5-(2-nitrooxy)-ethyl)-thiazol-2-yl]-butane (Pet-13),bis-[4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-yl]-diazene (Pet-102) and4,4′-dimethyl-5,5′-bis-(2-nitrooxy-ethyl)-[2,2′]bithiazolyl (Pet-118)(see, Table 1).

Further optionally, X can be a thiazole, such that the compound containstwo thiazole moieties attached therebetween. Such compounds may providefor additive advantageous effects of the thiazole residue, discussedhereinabove.

When X is a thiazole ring, the thiazole can be substituted ornon-substituted. When substituted, each substituent can be, for example,alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic, aryl,heteroaryl, amine, C-amide, N-amide, halide, acyl-halide, haloalkyl,sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, N-sulfonamide,S-sulfonamide, C-carboxylate, O-carboxylate, C-thiocarboxylate,O-thiocarboxylate, N-carbamate, O-carbamate, N-thiocarbamate,O-thiocarbamate, S-dithiocarbamate, N-dithiocarbamate, urea, thiourea,guanyl, guanidine and hydrazine, as these terms are defined hereinabove.

Alternatively, one or more substituents can be a moiety containing anNO-releasing group, as described hereinabove. Such compounds may providefor additive advantageous effects of both the thiazole residues and theNO-releasing groups.

Further optionally and preferably, X is a bioactive agent residue.

The phrase “bioactive agent” is used herein to describe an agent capableof exerting a beneficial activity in a biological system (e.g., a livingtissue or organ) of a subject. The beneficial activity includes, forexample, a therapeutic activity per se, reduction of adverse sideeffects induced by another moiety or agent, and/or targeting and/ortransportation of another moiety and/or agent towards a desiredbiological target.

The term “residue”, as used in this context of the present invention,refers herein to a major portion of a molecule, which is covalentlylinked to another molecule, herein the chemical moiety (e.g., athiamine-derived thiazole), or alternatively, is formed upon cleavage ofanother molecule.

Representative examples of bioactive agents that can be beneficiallyincorporated in the NO-donating compounds of the present inventioninclude, without limitation, drugs, inhibitors, co-factors, co-enzymes,amino acids, peptides, proteins, hydroxamic acid, nicotinic acid,nicotinamide, carnitine, beta carotene, bromelain, non-steroidalanti-inflammatory drugs (NSAIDs), anti-psychotic agents,anti-thrombogenic agents, anti-platelet agents, anti-coagulants,anti-diabetics, growth factors, statins, toxins, antimicrobial agents,analgesics, metabolite agents, anti-metabolic agents, vasoactive agents,vasodilator agents, prostaglandins, hormones, thrombin inhibitors,enzymes, oligonucleotides, nucleic acids, antisenses, antibodies,antigens, vitamins, immunoglobulins, cytokines, cardiovascular agents,chemotherapeutic agents, antioxidants, phospholipids, anti-proliferativeagents and heparins.

Non-limiting examples of non-steroidal anti-inflammatory drugs that canbe beneficially incorporated in the NO-donors of the present inventioninclude aspirin, celecoxib, diclofenac, diflunisal, etodolac,fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen,ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen,oxaprozin, oxyphenbutazone, phenylbutazone, piroxicam, rofecoxibsulindac and tolmetin.

Non-limiting examples of anti-psychotic agents that can be beneficiallyincorporated in the NO-donors of the present invention includealprazolam, amantadine, amitriptyline, amoxapine, benztropine,bupropion, buspirone, calcium carbimide, carbamazepine,chlordiazepoxide, chlorpromazine, citalopram, clomipramine, clonazepam,clozapine, desipramine, dextroamphetamine, diazapam, diphenhydramine,disulfiram, divalproex, doxepin, edronax, ethosuximide, fluoxetine,flupenthixol, fluphenazine, flurazepam, fluvoxamine, haloperidol,imipramine, lamotrigine, lithium, lorazepam, loxapine, maprotiline,mesoridazine, methylphenidate, moclobemide, nefazodone, nortriptyline,olanzapine, oxazepam, paroxetine, pemoline, perphenazine, phenelzine,phenytoin, pipotiazine, primidone, propranolol, protriptyline,quetiapine, reboxetine, risperidone, sabril, sertraline, temazepam,thioridazine, tranylcypromine, trazodone, triazolam, trifluoperazine,trihexyphenidyl, trimipramine, valproate, venlafaxine, verapamil,vigabatrin, zopiclone and zuclopenthixol.

Non-limiting examples of cardiovascular agents that can be beneficiallyincorporated in the NO-donors of the present invention includeadenosinea, alteplase, amiodarone, anagrelide, argatroban, atenolol,atorvastatin, benazepril, captopril, carvedilol, cerivastatin,clonidine, clopidrogel, diltiazem, enalapril, fluvastatin, fosinopril,gemfibrozil, hydrochlorothiazide, irbesartan, lisinopril, lovastatin,mibefradil, oprelvekin, pravastatin, prazosin, quinapril, ramipril,simvastatin, terazosin, valsartan and verapamil.

Non-limiting examples of metabolites that can be beneficiallyincorporated in the NO-donors of the present invention include glucose,urea, ammonia, tartarate, salicylate, succinate, citrate, nicotinateetc.

Non-limiting examples of anti-thrombogenic agents that can bebeneficially incorporated in the NO-donors of the present inventioninclude dipyridamole, tirofiban, aspirin, heparin, heparin derivatives,urokinase, rapamycin, PPACK (dextrophenylalanine proline argininechloromethylketone), probucol, and verapamil.

Non-limiting examples of chemotherapeutic agents that can bebeneficially incorporated in the NO-donors of the present inventioninclude amino containing chemotherapeutic agents such as daunorubicin,doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, anthracycline,mitomycin C, mitomycin A, 9-amino camptothecin, aminopertin,antinomycin, N⁸-acetyl spermidine,1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazine, bleomycin,tallysomucin, and derivatives thereof; hydroxy containingchemotherapeutic agents such as etoposide, camptothecin, irinotecaan,topotecan, 9-amino camptothecin, paclitaxel, docetaxel, esperamycin,1,8-dihydroxy-bicyclo[7.3.1]trideca-4-ene-2,6-diyne-13-one, anguidine,morpholino-doxorubicin, vincristine and vinblastine, and derivativesthereof, sulfhydril containing chemotherapeutic agents and carboxylcontaining chemotherapeutic agents.

Non-limiting examples of antimicrobial agents that can be beneficiallyincorporated in the NO-donors of the present invention include iodine,chlorhexidene, bronopol and triclosan.

Non-limiting examples of vitamins that can be beneficially incorporatedin the NO-donors of the present invention include vitamin A, thiamin,vitamin B₆, vitamin B₁₂, vitamin C, vitamin D, vitamin E, vitamin K,riboflavin, niacin, folate, biotin and pantothenic acid.

Non-limiting examples of anti-diabetics that can be beneficiallyincorporated in the NO-donors of the present invention include lipoicacid, acarbose, acetohexamide, chlorpropamide, glimepiride, glipizide,glyburide, meglitol, metformin, miglitol, nateglinide, pioglitazone,repaglinide, rosiglitazone, tolazamide, tolbutamide and troglitazone.

The conjugation of a bioactive agent to a group having an NO-releasinggroup attached thereto is highly beneficial since it may provide forcombined and even synergistic therapeutic effects of both theNO-releasing group and the bioactive agent.

These combined therapeutic effects are particularly advantageous whenthe bioactive agent is associated with NO-deficiency related impairment,namely, an administration of the bioactive agent to a subject, whichadversely causes NO-deficiency. Examples of such bioactive agents whichadversely cause NO-deficiency include, without limitation,anti-psychotic agents, cardiovascular agents and particularlynon-steroidal anti-inflammatory agents (NSAIDs).

Thus, according to a preferred embodiment of the present invention, thebioactive agent residue (X on Formula I above) is a non-steroidalanti-inflammatory drug residue.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used asanti-inflammatory, analgesic and antipyretic for the treatment of pain,fever, and inflammation. Chronic NSAID therapy effectively reduces thesymptoms of many painful arthritic syndromes, but is oftentimesassociated with adverse gastrointestinal (GI) complications [Cash, J.M.; Klippel, J. H. New Engl. J. Med., 1994, 330, 1368; Davies, N. M.,Wallace, J. L. J. Gastroenterol., 1997, 32, 127; Wallace, J.Gastroenterol., 1997, 112, 1000], as well as high blood pressure andheart diseases. At the tissue level, the most common clinicalmanifestation of NSAID-related GI damage is a combination ofgastroduodenal erosions and ulcerations often called NSAID-inducedgastropathy, affecting at least 25% of chronic NSAID patients.NSAID-induced gastropathy may limit long-term NSAID therapy and cause asignificant financial burden to the healthcare system.

In vivo NO generation has become the prime therapeutic target forreducing NSAID induced gastropathy associated with chronic NSAID use. Arecently published data have shown that NO-donors effectively reducegastric mucosal damage and may facilitate GI healing following chemicalinsult [Ko, J. K.; Cho, C. H. Inflamm. Res., 1999, 48, 471]. As firstconceptualized by Wallace and colleagues [Reuter, B., Wallace, J. L. Atherapeutic application of nitric oxide: GI-sparing NSAIDs. In: NitricOxide: A Modulator of Cell-Cell Interactions in the Microcirculation.(P. Kubes, ed.) R. G. Landes Company, 1995, pp. 157-168], modern drugdiscovery has focused on one general approach in an attempt to utilizethe therapeutic potential of NO against NSAID-induced gastric damage:covalent modification of NSAIDs with NO-releasing moieties [BrzozowskiT., et al., Dig Liver Dis. 2000 32(7), pp 583-94].

Due to the beneficial effect of compounds that can act as both NO-donorsand anti-inflammatory agents, delineated above, the present inventorshave designed and successfully prepared representative thiazole-basedcompounds, according to this embodiment of the present invention, whichhave a NSAID residue attached thereto (for example, as X in Formula Iabove). These include2-[1-(6-methoxy-naphthalen-2-yl-ethyl]-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet 17), wherein X is a naproxen residue,2-[1-(4-isobutyl-phenyl)-ethyl]-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-66) wherein X is an ibuprofen residue and acetic acid2-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl ester (Pet-116)wherein X is an aspirin residue (see, Tables 1 and 2).

As is demonstrated in the Examples section that follows, these novelcompounds exert high NO-releasing efficacy, are non-tolerance inducingand therefore further exert the protective effect required to improvethe safety and pharmacokinetic profile of the NSAID agent residueattached thereto.

The combined therapeutic effects mentioned above are furtheradvantageous when the condition that is treatable by the bioactive agentis further associated with NO-deficiency and/or may lead to clinicalmanifestations in which elevating the NO level is beneficial. An exampleof such a condition is diabetes.

Thus, according to preferred embodiments of the present invention, thebioactive agent residue (X in Formula I above) is an anti-diabetic agentresidue, as is detail hereinabove.

The link between diabetes mellitus and vascular diseases has been wellestablished in many studies, indicating that the main etiology formortality and a great percent of morbidity in patients with diabetesmellitus is atherosclerosis (Calles-Escandon, J. and M. Cipolla (2001),Endocr. Rev. 22(1): 36-52). The reciprocity between insulin and NOproductions, as well as blood-circulation irregularities and systemiclow NO-levels that are typically associated with diabetes, accentuatethe interrelationship between diabetes of all types and NO deficiency.

Thus, it was found that narrowing of blood vessels due to increasedsynthesis of type IV collagen by vascular cells adjacent to the membraneoccurs in response to hyperglycaemia, hyperinsulinaemia, and otherfactors associated with diabetes (Sharma K. et al., (1996), Diabetes,45: 522-30). There are also evidences that the vasodilatation activityof insulin may be partly mediated by local and systemic NO levels, andvice versa (Scherrer U. et al., (1994) J. Clin Invest; 94: 2511-15).

In addition, NO deficiency is prevalent in people with diabetes. BothType I and Type II diabetic patients have a reduced ability to generateNO from L-arginine, reflected in part by direct measurements of plasmanitrate and nitrite levels. This is mainly attributed to themalfunctioning of NOS, which, in turn, results from reduction in theelimination of a natural inhibitor of NOS (asymmetrical dimethylarginine, ADMA) due to reduced kidney function, impaired bloodcirculation and hence inadequate oxygen levels which leads to impairedNO-generation by NOS, and an acidic pH which adversely affect NOSactivity, all are observed in patients having diabetes.

Hence, since it is well established that elevating the NO level whiletreating a patient having diabetes is known to be beneficial, thepresent inventors have designed and successfully prepared representativethiazole-based compounds, according to this embodiment of the presentinvention, which have an anti-diabetic agent residue attached thereto(for example, as X in Formula I above). These include2-(4-[1,2]dithiolan-3-yl-butyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-67) and 5-[1,2]dithiolan-3-yl-pentanoic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amide (Pet-151), in which Xis a lipoic acid residue (see, Tables 1 and 2). As is well known in theart, lipoic acid is a therapeutically active agent that is used as afood supplement to assist in the combat against diabetes.

Each of the bioactive agent residues described above (for example, X inFormula I above) can be attached to the chemical moiety (e.g., thethiamine-derived thiazole ring) either directly or indirectly. Whenattached indirectly, the bioactive agent is attached to the chemicalmoiety (e.g., the thiazole ring) via a linking moiety, represented, forexample, as A in Formula I above.

The linking moiety (e.g., A in Formula I) can be, for example, alkenyl,alkoxy, alkyl, alkynyl, amine, amine-oxide, aryl, aryloxy, azo, borate,C-amide, carbonyl, C-carboxylate, C-thiocarboxylate, cycloalkyl, diazo,disulfide, guanidine, guanyl, haloalkyl, heteroalicyclic, heteroaryl,hydrazine, N-amide, N-carbamate, N-dithiocarbamate, nitro,N-sulfonamide, N-thiocarbamate, O-carbamate, O-carboxylate,O-thiocarbamate, O-thiocarboxylate, oxime, oxygen, peroxo, phosphate,phosphine-oxide, phosphine-sulfide, phosphinyl, phosphite, phosphonate,pyrophosphate, S-dithiocarbamate, silaza, silicate, siloxy, silyl,S-sulfonamide, sulfate, sulfite, sulfonate, sulfoxide, sulfur,thioalkoxy, thioaryloxy, thiocarbonyl, thiophosphate, thiosulfate,thiosulfite, thiourea, triphosphate, urea, a biocleavable moiety and anycombination thereof, or absent.

According to a preferred embodiment of the present invention, thelinking moiety (e.g., A in Formula I) is a biocleavable moiety.

As used herein, the phrase “biocleavable moiety” describes a chemicalmoiety, which undergoes cleavage in a biological system such as, forexample, the digestive system of an organism or an enzymatic system in aliving cell. Representative examples of biocleavable moieties include,without limitation, amides, carboxylates, carbamates, phosphates,hydrazides, thiohydrazides, disulfides, epoxides, peroxo andmethyleneamines. Such moieties are typically subjected to enzymaticcleavages in a biological system, by enzymes such as, for example,hydrolases, amidases, kinases, peptidases, phospholipases, lipases,proteases, esterases, epoxide hydrolases, nitrilases, glycosidases andthe like.

As used herein, the term “hydrazide” describes a —C(═O)—NR′—NR″R′″ endgroup or a —C(═O)—NR′—NR″— linking group, as these phrases are definedhereinabove, where R′, R″ and R′″ are as defined herein.

As used herein, the term “thiohydrazide” describes a —C(═S)—NR′—NR″R′″end group or a —C(═S)—NR′—NR″— linking group, as these phrases aredefined hereinabove, where R′, R″ and R′″ are as defined herein.

As used herein, the term “epoxide” describes a

end group or a

linking group, as these phrases are defined hereinabove, where R′, R″and R′″ are as defined herein.

As used herein, the term “methyleneamine” describes an—NR′—CH₂—CH═CR″R′″ end group or a —NR′—CH₂—CH═CR″— linking group, asthese phrases are defined hereinabove, where R′, R″ and R′″ are asdefined herein.

As discussed hereinabove, some preferred NO-donors according to thepresent invention have a bioactive agent residue attached thereto andtherefore offer exceptional advantages due to the dual functionalitythereof (elevating the NO level by an NO-releasing group and exerting abeneficial activity by the bioactive agent). Incorporation of abiocleavable moiety which links between the bioactive agent residue andthe chemical moiety attached to the NO-releasing group (e.g., a nitratedthiamine-derived thiazole ring) in such compounds provides for a releaseof the bioactive agent in a biological system and thus may improve thebiological activity of both the NO-releasing part of the compound andthe bioactive agent.

Thus, according to another preferred embodiment of the presentinvention, the NO-donating compounds according to the present inventioninclude a bioactive agent (e.g., X in Formula I), which is attached tothe chemical moiety via a biocleavable moiety (e.g., A in formula I).

As is demonstrated in the Examples section that follows, representativeexamples of thiamine-derived thiazole-based NO-donating compounds inwhich A is a biocleavable moiety and X is a bioactive residue have beensuccessfully prepared. These include, for example,4-[1,2]dithiolan-3-yl-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-butyramide(Pet-151) wherein A is an amide and X is a residue of5-[1,2]dithiolan-3-yl-pentanoic acid, also known as lipoic acid, andhave been suggested as a therapeutic and prophylactic treatment of manyage-related diseases, from heart disease and stroke to diabetes andcataracts, 4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acidN′-phthalazin-1-yl-hydrazide (Pet-153) wherein A is a hydrazide and X isphthalazine-1-yl,N-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-nicotinamide (Pet-154)wherein A is an amide and X is a nicotinic acid residue,allyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine (Pet-155)wherein A is an amine and X is allyl (H₂C═CH—CH₂—),N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-1-oxy-nicotinamide(Pet-156) wherein A is an amide and X is an oxidized nicotinic acidresidue (pyridine 1-oxide-3-yl), hexadecanoic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amide (Pet-158) wherein Ais an amide and X is 1-pentadecanyl (fatty acid), and10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-ylester (Pet-164) wherein A is an amide and X is a hormone residue.

Other exemplary thiamine-derived thiazole-based NO-donating compounds inwhich A is a biocleavable moiety which have been successfully preparedinclude4-acetylamino-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-benzamide(Pet-157); pyrrolidine-2-carboxylic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amide (Pet-160);2,6-difluoro-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-benzamide(Pet-161);2-(2,4-dichloro-phenyl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide(Pet-162); and2-(2,4-dichloro-phenoxy)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide(Pet-163).

As is further discussed hereinabove, NO-donors according to the presentinvention in which the bioactive agent residue is a NSAID residue arehighly beneficial and hence attaching the NSAID residue to the thiazolering that contains an NO-releasing moiety would be highly advantageous.Thus, in another preferred embodiment of the present invention, A is abiocleavable moiety and X is a NSAID residue.

As is demonstrated in the Examples section that follows, representativeexamples of compounds in which A is a biocleavable moiety and X is aNSAID residue have been successfully prepared. These include2-(6-methoxy-naphthalen-2-yl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propionamide(Pet-150), where A is an amide and X is a naproxen residue,2-(4-isobutyl-phenyl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propionamide(Pet-152) where A is an amide and X is an ibuprofen residue and aceticacid 2-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylcarbamoyl]-phenylester (Pet-159) wherein A is an amide and X is an Aspirin residue(2-acetobenzene-1-yl).

The chemical structures of preferred compounds according to thisembodiment of the present invention are set forth in Table 1 below. Alist of preferred compounds according to the present invention is setforth in Table 2 below.

The present invention further encompasses any pharmaceuticallyacceptable salts of the NO-donating compounds described herein.

The phrase “pharmaceutically acceptable salt” refers to a chargedspecies of the parent compound and its counter ion, which is typicallyused to modify the solubility characteristics of the parent compoundand/or to reduce any significant irritation to an organism by the parentcompound, while not abrogating the biological activity and properties ofthe administered compound.

The present invention further encompasses prodrugs, solvates andhydrates of the NO-donating compounds described herein.

As used herein, the term “prodrug” refers to an agent, which isconverted into the active compound (the active parent drug) in vivo.Prodrugs are typically useful for facilitating the administration of theparent drug. They may, for instance, be bioavailable by oraladministration whereas the parent drug is not. The prodrug may also haveimproved solubility as compared with the parent drug in pharmaceuticalcompositions. Prodrugs are also often used to achieve a sustainedrelease of the active compound in vivo. An example, without limitation,of a prodrug would be the NO-donating compound, having one or morecarboxylic acid moieties, which is administered as an ester (the“prodrug”). Such a prodrug is hydrolysed in vivo, to thereby provide thefree compound (the parent drug). The selected ester may affect both thesolubility characteristics and the hydrolysis rate of the prodrug.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the NO-donating compound) and a solvent, whereby the solventdoes not interfere with the biological activity of the solute. Suitablesolvents include, for example, ethanol, acetic acid and the like.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

Chemical Syntheses:

Further according to the present invention, there is provided a processof preparing the novel NO-donors of the present invention describedhereinabove, and particularly the preferred thiazole-based NO-donorsdescribed hereinabove. The process is effected as follows:

As a starting material, a suitable thioamide, which includes themoieties A and X described hereinabove and is represented by the generalFormula II below is provided.

Some suitable thioamides according to the present invention arecommercially available or can be readily prepared. These include,without limitation, N,N-dimethylthiourea, thiobenzamide, thiourea,N-allylthiourea, acetylthiourea, N-ethylthiourea, N,N-dimethylthiourea,alpha-naphthylthiourea, 1-[(3,5-bis-trifluoromethyl)phenyl]thiourea anddithiooxalamide.

Other thioamides can be readily synthesized by converting acorresponding amide thereto. The selected amide preferably has thegeneral formula V below.

Some suitable amides according to the present invention are commerciallyavailable or can be prepared according to procedures known in the art.These include, without limitation, propionamide, acetamide,isobutyramide, L-(−)-lactamide, trifluoroacetamide, carbamic acid methylester, hexanedioic acid diamide, piperidine-4-carboxylic acid amide,thionicotinamide, naproxenamide, 4-(trifluoromethyl)-thiobenzamide,azodicarbonamide, 2-(4-isobutyl-phenyl)-propionamide, isonicotinamide,2,2,2-trifluoroacetamide, glycinamide, 4-aminobenzamide,2,3,4,5,6-pentafluorobenzamide, 2-aminobenzamide, ethyloxamate,2,6-difluorobenzamide, N-phenylurea, 2,4-dichlorophenylacetamide,2,4-dichlorophenoxyacetmide, 2-phenylbutyamide, azodicarbonamide,3,5-difluorobenzamide, DL-lipoamide, Rubeanic acid, adpamide,aalonamide, acrylamide and 2-hydroxy-benzamide.

Converting the selected amide to a thioamide is preferably effected byreacting the thioamide with a thiolating agent. The thiolating agent canbe any of those known in the art. Preferably, the thiolating agent isphosphorous pentasulfide.

The thioamide is then reacted with a suitable reactive compound, tothereby form, via a typical condensation reaction, a derivatizedthiazole ring.

Suitable reactive compounds for this purpose are those having thegeneral Formula III:

in which B and Z are as defined hereinabove, L is a leaving group and Wis a pre-nitratable group.

As used herein, the phrase “leaving group” describes a labile atom,group or chemical moiety that readily undergoes detachment from anorganic molecule during a chemical reaction, while the detachment isfacilitated by the relative stability of the leaving atom, group ormoiety thereafter. Typically, any group that is the conjugate base of astrong acid can act as a leaving group. Representative examples ofsuitable leaving groups according to the present invention thereforeinclude, without limitation, acetate, tosylate, hydroxy, thiohydroxy,alkoxy, halide, amine, azide, cyanate, thiocyanate, nitro and cyano.Preferably, the leaving group L is a halide, and most preferably it ischloride.

The phrase “pre-nitratable group”, as used herein, describes a chemicalmoiety that can be converted to a nitratable group, as this term isdefined hereinbelow. The conversion can be effected either spontaneouslyduring the condensation reaction described above, or non-spontaneously,via an additional reaction. The pre-nitratable group can be, forexample, a protected nitratable group, which undergoes deprotection andthus converted to a nitratable group during a reaction. Preferably, thepre-nitratable group according to the process of the present inventionis spontaneously converted into a nitratable group during the reactionof the reactive compound with the thioamide.

The reactive compound having the Formula III is therefore selected so asto have the desired B and Z moieties in the resulting NO-donatingcompound and a pre-determined pre-nitratable group, which is thereafterconverted into the desired NO-releasing group in the resulting compound,as is detailed hereinunder.

An example of a readily synthesizable reactive compound that can beadvantageously used in the process of the present invention is5-acetoxy-3-chloro-2-pentanone.

Upon reacting the thioamide and the reactive compound having FormulaIII, a derivatized thiazole, having the general Formula IV below, isobtained.

In general Formula IV, A, X, Z and B are as defined hereinabove and U isa nitratable group.

As used herein, the phrase “nitratable group” describes a chemicalmoiety, which can be readily converted to a NO-releasing group, as thisphrase is defined hereinabove, preferably by reacting it with anitrating agent.

Representative examples of nitratable groups according to the presentinvention are hydroxyl and thiohydroxyl. The hydroxyl or thiohydroxylgroup can be reacted with nitric acid, any derivative thereof or anycompound that includes a nitric acid residue, preferably under acidicconditions, to thereby produce an NO-releasing group.

In cases where the nitratable group according the present invention ishydroxy, or a thiohydroxy group the pre-nitratable group in Formula IIIabove can be, for example, a carboxylate, as this term is definedhereinabove, such as acetate. Under suitable reaction conditions, theacetate is spontaneously converted to hydroxy during the reactionbetween the thioamide and the reactive compound of Formula III.

The derivatized thiazole having general Formula IV is then reacted so asto convert the nitratable group into a NO-releasing group. Thisconversion is preferably effected by reacting the thiazole derivativewith a nitrating agent, which contains the NO-releasing group.

Suitable nitrating agents include, for example, nitric acid, which isused to provide an —ONO₂ NO-releasing group, and nitric oxide andoxygen, which are used to provide NONOates.

Exemplary conditions and procedures, which can be used in the processdescribed above, are described in detail in the Examples section thatfollows.

As is further shown in the Examples section that follows, using theprocess according to this aspect of the present invention a variety ofNO-donors according to the present invention have been successfullyprepared and characterized. These include, for example,2-Ethyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-2),2,4-Dimethyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-3),2-Isopropyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-4),4-Methyl-5-(-2-nitrooxy-ethyl)-2-(1-nitrooxy-ethyl-thiazole (Pet-5),4-Methyl-5-(nitrooxy-ethyl)-2-trifluoromethyl-thiazole (Pet-6),dimethyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine (Pet-7),4-methyl-5-(2-nitrooxy-ethyl)-2-phenyl-thiazole (Pet-8),2-methoxy-4-methyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-9),4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-ylamine (Pet-10),4-[4-methyl-5-(2-nitrooxy-ethyl)thiazole-2-yl]-piperidine (Pet-11),3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-yl]-pyridine (Pet-12),1,4-Bis-[4-Methyl-5-(2-nitrooxy)-ethyl)-thiazol-2-yl]-Butane (Pet-13),2-[1-(6-methoxy-naphthalen-2-yl-ethyl]-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-17),4-methyl-5-(2-nitrooxy-ethyl)-2-(4-trifluoromethyl-phenyl)-thiazole(Pet-59),2-[1-(4-isobutyl-phenyl)-ethyl]-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-66), acetic acid2-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl ester (Pet-116),4,4′-dimethyl-5,5′-bis-(2-nitrooxy-ethyl)-[2,2′]bithiazolyl (Pet-118),2-(3,5-difluoro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-181)4-chloro-1-methoxy-benzen-2-yl,2-(5-chloro-2-methoxy-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-182), N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-guanidine(Pet-183),N-(2-chloro-benzylidene)-N′-[4-methyl-5-(nitrooxy-ethyl)-thiazol-2-yl]-hydrazine(Pet-184),(4-chlorophenyl)-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine(Pet-185) and(3,5-dichloro-phenyl)-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine(Pet-186) (see, Table 1).

As is discussed in detail hereinabove, some of the presently preferredcompounds according to the present invention have a biocleavable moiety(A) linking between X and the thiazole ring (see, Formula I above). Dueto the cleavable nature of the biocleavable moiety, a different processhas been developed and successfully used for preparing such compounds.

Hence, according to still another aspect of the present invention thereis provided a process of preparing the NO-donors of the presentinvention described hereinabove and presented in Formula I above, inwhich A is a biocleavable moiety, as defined hereinabove. The process,according to this aspect of the present invention is effected asfollows:

Using the synthetic strategy described above for obtaining the thiazolederivative having the general Formula IV, suitable thioamides (FormulaII above) and reactive compounds having Formula III above are reacted soas to provide a derivatized thiazole having the general Formula VI:

wherein B, U and Z are as defined hereinabove, and Q is a first reactivegroup, as is defined and detailed hereinbelow.

Such a thiazole derivative is preferably obtained by selecting an amideor a thioamide, which has the general Formula VIII or IX, respectively:

The first reactive group Q in the amide or thioamide starting materialsis preferably protected by a suitable protecting group when thematerials are reacted to form the derivatized thiazole (Formula VI) andis thereafter deprotected. Any of the commonly used protecting groupscan serve as protecting groups in this embodiment of the presentinvention, depending on the selected reactive group and the reactionconditions.

A reactive derivative of X, which has the general Formula VII below, isfurther provided.X—K  Formula VII

In Formula VII, X is as defined above and K is a second reactive group,as is defined hereinbelow.

The compound of Formula VII can be a commercially available compound orcan be prepared using commonly known synthetic strategies, depending onthe selected X and K.

As used herein, the phrase “reactive group”, which refers to both thefirst and the second reactive groups, describes a chemical moiety thatis capable of undergoing a chemical reaction that typically leads to abond formation. The bond, according to the present invention, ispreferably a covalent bond. Chemical reactions that lead to a bondformation include, for example, nucleophilic and electrophilicsubstitutions, nucleophilic and electrophilic addition reactions,cycloaddition reactions, rearrangement reactions and any other knownorganic reactions that involve a reactive group.

Representative examples of suitable reactive groups according to thepresent invention include, without limitation, amine, halide,acyl-halide, sulfonate, sulfoxides, phosphate, hydroxy, alkoxy, aryloxy,thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, isocyanate,sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate,O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide,N-amide, guanyl, guanidine and hydrazine, as these terms are definedhereinabove.

The first and the second reactive groups are preferably selected asbeing capable to react one with the other, to thereby form the desiredbiocleavable moiety.

Thus, for example, amine and hydroxy are selected as the first andsecond reactive groups or, vice versa, as the second and first reactivegroups, so as to form an amide as the biocleavable moiety; a carboxylateor acyl-halide and hydroxy are selected so as to form a carboxylate-typebiocleavable moiety; two thiohydroxy groups are selected so as to form adisulfide biocleavable moiety, an isocyanate and a hydroxy are selectedso as to form a carbamate-type biocleavable moiety; and a hydrazine anda carboxylic acid are selected so as to form a hydrazide-typebiocleavable moiety.

The phrase “carboxylate-type biocleavable moiety” includes a carboxylatelinking group, as defined hereinabove, and any derivative thereof, suchas, for example, thiocarboxylate.

The phrase “carbamate-type biocleavable moiety” includes a carbamatelinking group, as defined hereinabove, and any derivative thereof, suchas, for example, thiocarbamate.

The phrase “hydrazide-type biocleavable moiety” includes a hydrazidelinking group, as defined hereinabove, and any derivative thereof, suchas, for example, thiohydrazide.

The thiazole derivative and the compound of Formula VII are thenreacted, under suitable conditions which enable the formation of a bondbetween the first and the second reactive groups, to thereby provide aderivatized thiazole having the general Formula IV, as describedhereinabove.

The nitratable group in the derivatized thiazole is then converted, asdescribed hereinabove, to the NO-releasing group, so as to provide thedesired NO-donor according to this embodiment of the present invention.

As is demonstrated in the Examples section, using the process accordingto this aspect of the present invention, a variety of NO-donorsaccording to the present invention, which have a biocleavable moiety,have been successfully prepared. These include, for example,2-(6-methoxy-naphthalen-2-yl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propionamide(Pet-150),4-[1,2]dithiolan-3-yl-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-butyramide(Pet-151),2-(4-Isobutyl-phenyl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propionamide(Pet-152), 4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acidN′-phthalazin-1-yl-hydrazide (Pet-153),N-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-nicotinamide (Pet-154),allyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine (Pet-155),N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-1-oxy-nicotinamide(Pet-156),4-acetylamino-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-benzamide(Pet-157), hexadecanoic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amide (Pet-158), aceticacid 2-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylcarbamoyl]-phenylester (Pet-159), Pyrrolidine-2-carboxylic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amide (Pet-160),2,6-difluoro-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-benzamide(Pet-161),2-(2,4-dichloro-phenyl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide(Pet-162),2-(2,4-dichloro-phenoxy)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide(Pet-163) and 4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acid10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-ylester (Pet-164) (see, Table 1).

As is demonstrated in the Examples section that follows, the compoundsdescribed above were found to be highly active NO-donating agents. In aseries of both in vitro and in vivo assays, the NO-donors according tothe present invention were found highly efficient in inducingvasorelaxation, while no tolerance phenomenon was observed upon repeatedadministration thereof.

These results indicate that the NO-donors of the present invention canbe beneficially used in the treatment of medical conditions associatedwith NO.

Hence, further according to the present invention there is provided amethod of treating or preventing a medical condition in which modulatingan NO level is beneficial. The method, according to this aspect of thepresent invention, is effected by administering to a subject in needthereof a therapeutically effective amount of one or more of theNO-donating compounds described above.

As used herein, the phrase “therapeutically effective amount” describesan amount of the compound being administered which will relieve to someextent one or more of the symptoms of the disorder being treated, as isfurther detailed hereinunder.

An exemplary therapeutically effective amount of an NO-donor of thepresent invention ranges between about 0.01 mg/kg body and about 5 mg/kgbody. It should be noted herein that GTN is presently marketed as unitdosage forms of 5 mg, which is equivalent to about 0.07 mg/kg body,assuming an average human body weight of about 70 kg. As is demonstratedin the Example section that follows, the NO-donors of the presentinvention are at least as effective as GTN in treating hypertension inanimals, and in most cases show superior efficacy as compared with GTN.A typical unit dosage from of the NO-donating compounds of the presentinvention can therefore be equal to or lower than 0.07 mg/kg body.

As used herein the term “about” refers to ±10%.

As is delineated hereinabove, inadequate somatic NO levels areassociated with various biological dysfunctions, which typically resultfrom or lead to adverse decrease in the somatic NO levels. Administeringan NO-donating compound to subjects that suffer from such inadequatesomatic NO levels therefore ameliorate the biological dysfunction itselfor its symptoms. Thus, preferably, medical conditions, which arebeneficially treatable by the method according to this aspect of thepresent invention, are those in which elevating the NO-level in asubject is beneficial.

Non-limiting examples of medical conditions in which modulating, andpreferably elevating, the NO level is beneficial include cardiovasculardiseases or disorders, gastrointestinal diseases or disorders,inflammatory diseases or disorders, respiratory diseases or disorders,central nervous system diseases or disorders, neurodegenerative diseasesor disorders, psychiatric diseases or disorders, bloodpressure-associated diseases or disorders, coronary artery diseases ordisorders, atherosclerosis, cholesterol level-associated diseases ordisorders, arterial thrombotic diseases or disorders, a heart failure, astroke, a septic shock, NSAID-induced gastric diseases or disorders,inflammatory bowel diseases or disorders, ischemic renal diseases ordisorders, peptic ulcer, diabetes, pulmonary hypertension, sickle cellanemia, an asthma, chronic obstructive pulmonary disease, dementia,epilepsy, neuroinflammatory diseases or disorders, trauma, multiplesclerosis, erectile dysfunction, other male and female sexualdysfunctions and age-related diseases or disorders.

As is discussed hereinabove, NO-donating compounds according to thepresent invention, and particularly those that include a NSAID residue,are highly beneficial in treating inflammation. These compounds can thusbe efficiently utilized to treat a variety of diseases and disordersassociated with inflammation, such as, for example, reperfusion injuryof an ischemic organ, e.g., reperfusion injury of the ischemic,myocardium, myocardial infarction, inflammatory bowel disease,rheumatoid arthritis, osteoarthritis, hypertension, psoriasis, organtransplant rejections, organ preservation, impotence, radiation-inducedinjury, asthma, atherosclerosis, thrombosis, platelet aggregation,metastasis, influenza, stroke, burns, trauma, acute pancreatitis,pyelonephritis, hepatitis, autoimmune diseases, insulin-dependentdiabetes mellitus, diabetes type II, disseminated intravascularcoagulation, fatty embolism, Alzheimer's disease, Parkinson's disease,multiple sclerosis, neonate-, infantile- and adult-respiratory diseases,carcinogenesis, hemorrhages in neonates, cerebral vascular disorders andother pathological conditions.

As is further discussed hereinabove, NO-donating compounds according tothe present invention, and particularly those that include ananti-diabetic agent residue, are highly beneficial in treating diabetesof all types. These compounds can thus be efficiently utilized to treatfor, example, diabetes mellitus, diabetes Type I juvenile) andnon-insulin dependent diabetes Type II.

The NO-donors according to the present invention can be administeredorally, rectally, intravenously, intraventricularly, topically,intranasally, intraperitoneally, intestinally, parenterally,intraocularly, intradermally, transdermally, subcutaneously,intramuscularly, transmucosally, by inhalation and/or by intrathecalcatheter. Preferably, the NO-donors, according to the present invention,are administered orally or intravenously, and optionally topically,transdermally or by inhalation, depending on the condition and thesubject being treated.

The method, according to this aspect of the present invention, canoptionally be effected by co-administering to the subject an additionalactive agent for treating the medical condition. The additional activeagent can be co-administered prior to, concomitantly or subsequent toadministering the compound of the present invention.

The additional active agent can be, for example, any of the agents knownfor treating the medical conditions described above such as, but notlimited to, cardiovascular agents, NSAIDs, anti-psychotic agents,anti-thrombogenic agents, anti-platelet agents, anti-coagulants,antimicrobial agents, analgesics, metabolite agents, anti-metabolicagents, vasoactive agents, vasodilator agents, chemotherapeutic agents,antioxidants, phospholipids, anti-proliferative agents, anti-diabetics,heparins and the like.

The NO-donors of the present invention, alone or in combination with anyother active agents, according to this aspect of the present invention,can be administered either per se, or as a part of a pharmaceuticalcomposition.

Hence, according to still another aspect of the present invention, thereare provided pharmaceutical compositions, which comprise one or more ofthe NO-donors described above and a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the NO-donors described herein, with other chemicalcomponents such as pharmaceutically acceptable and suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare: propylene glycol, saline, emulsions and mixtures of organicsolvents with water, as well as solid (e.g., powdered) and gaseouscarriers.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the NO-donors intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the NO-donors of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline bufferwith or without organic solvents such as propylene glycol, polyethyleneglycol.

For transmucosal administration, penetrants are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the NO-donors of the invention can beformulated readily by combining the NO-donors with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable theNO-donors of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for oral ingestion by a patient. Pharmacological preparations for oraluse can be made using a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active NO-donors doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theNO-donors may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the NO-donors for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation (which typically includes powdered, liquified and/orgaseous carriers) from a pressurized pack or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the NO-donors and a suitable powder base suchas, but not limited to, lactose or starch.

The NO-donors described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the NO-donors preparation in water-soluble form.Additionally, suspensions of the NO-donors may be prepared asappropriate oily injection suspensions and emulsions (e.g.,water-in-oil, oil-in-water or water-in-oil in oil emulsions). Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents, which increase the solubility ofthe NO-donors to allow for the preparation of highly concentratedsolutions.

Alternatively, the NO-donors may be in powder form for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The NO-donors of the present invention may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofNO-donors effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any NO-donors used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromactivity assays in animals. For example, a dose can be formulated inanimal models to achieve a circulating concentration range that includesthe IC₅₀ as determined by activity assays (e.g., the concentration ofthe test NO-donors, which achieves a half-maximal reduction of the meanarterial blood pressure). Such information can be used to moreaccurately determine useful doses in humans.

As is demonstrated in the Examples section that follows, atherapeutically effective amount for the NO-donors of the presentinvention may range between about 0.05 mg/kg body and about 5 mg/kgbody.

Toxicity and therapeutic efficacy of the NO-donors described herein canbe determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the EC₅₀, the IC₅₀ and the LD₅₀ (lethaldose causing death in 50% of the tested animals) for a subject NO-donor.The data obtained from these activity assays and animal studies can beused in formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thedesired effects, termed the minimal effective concentration (MEC). TheMEC will vary for each preparation, but can be estimated from in vitrodata; e.g., the concentration necessary to achieve 50-90% vasorelaxationof contracted arteries. Dosages necessary to achieve the MEC will dependon individual characteristics and route of administration. HPLC assaysor bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition described hereinabove, with course of treatment lasting fromseveral days to several weeks or until cure is effected or diminution ofthe disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA (the U.S. Food and DrugAdministration) approved kit, which may contain one or more unit dosageforms containing the active ingredient. The pack may, for example,comprise metal or plastic foil, such as, but not limited to a blisterpack or a pressurized container (for inhalation). The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accompanied by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert. Compositions comprising a NO-donors ofthe invention formulated in a compatible pharmaceutical carrier may alsobe prepared, placed in an appropriate container, and labeled fortreatment of an indicated condition or diagnosis, as is detailedhereinabove.

Thus, according to an embodiment of the present invention, depending onthe selected components of the NO-donors, the pharmaceuticalcompositions of the present invention are packaged in a packagingmaterial and identified in print, in or on the packaging material, foruse in the treatment of a condition in which modulating an NO level isbeneficial, as described hereinabove.

The NO-donors of the present invention can be further beneficiallyutilized as active substances in various medical devices.

Hence, according to an additional aspect of the present invention thereis provided a medical device which includes one or more of theNO-donating compounds of the present invention, described hereinabove,and a delivery system configured for delivering the NO-donatingcompound(s) to a bodily site of a subject.

The medical devices according to the present invention are thereforeused for delivering to or applying on a desired bodily site theNO-donating compounds of the present invention. The NO-donatingcompounds can be incorporated in the medical devices either per se or asa part of a pharmaceutical composition, as described hereinabove.

As used herein, the phrase “bodily site” includes any organ, tissue,membrane, cavity, blood vessel, tract, biological surface or muscle,which delivering thereto or applying thereon the compounds of thepresent invention is beneficial.

Exemplary bodily sites include, but are not limited to, the skin, adermal layer, the scalp, an eye, an ear, a mouth, a throat, a stomach, asmall intestines tissue, a large intestines tissue, a kidney, apancreas, a liver, the digestive system, the respiratory tract, a bonemarrow tissue, a mucosal membrane, a nasal membrane, the blood system, ablood vessel, a muscle, a pulmonary cavity, an artery, a vein, acapillary, a heart, a heart cavity, a male or female reproductive organand any visceral organ or cavity.

The medical devices according to this aspect of the present inventioncan be any medical device known in the art, including those defined andclassified, for example, by the FDA and specified inhttp://www.fda.gov/cdrh/devadvice/313.html (e.g., Class I, II and III),depending e.g., on the condition and bodily site being treated.

Thus, for example, in one embodiment of this aspect of the presentinvention, the medical device comprises a delivery system that isconfigured to deliver the compound by inhalation. Such inhalationdevices are useful for delivering the NO-donating compounds of thepresent invention to, e.g., the respiratory tract.

The delivery system in such medical devices may be based on any ofvarious suitable types of respiratory delivery systems which aresuitable for administering a therapeutically effective dose of thecompound of the present invention to a subject. The inhalation devicemay be configured to deliver to the respiratory tract of the subject,preferably via the oral and/or nasal route, the compound in the form ofan aerosol/spray, a vapor and/or a dry powder mist. Numerous respiratorysystems and methods of incorporating therapeutic agents therein, such asthe NO-donating compounds of the present invention, suitable forassembly of a suitable inhalation device are widely employed by theordinarily skilled artisan and are extensively described in theliterature of the art (see, for example to U.S. Pat. Nos. 6,566,324,6,571,790, 6,637,430, and 6,652,323; U.S. Food & Drug Administration(USFDA) Center For Drug Evaluation and Research (CDER);http://www.mece.ualberta.ca/arla/tutorial.htm).

The respiratory delivery system may thus be, for example, an atomizer oraerosol generator such as a nebulizer inhaler, a dry powder inhaler(DPI) and a metered dose inhaler (MDI), an evaporator such as anelectric warmer and a vaporizer, and a respirator such as a breathingmachine, a body respirator (e.g., cuirass), a lung ventilator and aresuscitator.

An exemplary medical device according to this embodiment of the presentinvention is a metered-dose inhaler (MDI). An MDI typically discharge ameasured amount of a therapeutic agent from a pressurized canister (forexample, Serevent® Inhalational Aerosol) using a compressed propellantgas. Typically, a human individual self-administers a therapeutic agentvia an MDI by applying pressure to a trigger on the MDI so as to delivera “burst” of a mixture of propellant and medicament into the mouthduring an inhalation, the propelling “burst” being provided by thepressure within the canister. Suitable formulations for MDIadministration of a therapeutic agent include a solution or suspensionof the therapeutic agent in a liquefied propellant (e.g.,chlorofluorocarbons and hydrofluoroalkanes). A suitable formulation forMDI administration can include, for example, from about 0.01% to about5% by weight of the NO-donating compound, from about 0% to about 20% byweight ethanol, and from about 0% to about 5% by weight surfactant, withthe remainder being the propellant. For examples of processes ofpreparing respirable particles, and formulations and devices suitablefor inhalation-dosing see, for example, U.S. Pat. Nos. 6,268,533,5,983,956, 5,874,063, and 6,221,398; and PCT Publication Nos. WO99/55319 and WO 00/30614. MDIs are advantageous in cases where an easilyportable hand-held device is desired. Conventional MDIs can be modifiedso as to increase the ability to obtain repeatable dosing by utilizingtechnology which measures the inspiratory volume and flow rate of asubject (refer, for example, to U.S. Pat. Nos. 5,404,871 and 5,542,410).Other types of MDI may deliver the therapeutic agent in a solid state,i.e., as a fine powder of respirable particle size combined with agaseous propellant.

An example of a typical hand-held MDI is shown in FIG. 34. Metered-doseinhaler 10 is composed of a pressurized cartridge 12 connected to aspray-nozzle 14 via housing 16. Pressurized cartridge 12 contains theliquid formulation 18, which includes a mixture of powdered or liquefiedpropellant and the NO-donating compound according to the presentinvention; and a pressurized propellant vapor 20. For administration ofthe metered dose, the mouth of the subject is sealed around a mouthpiece22, and pressurized cartridge 12 and housing 16 are squeezed togethermanually by the subject concomitantly with inhalation by the subject.The squeezing allows pressurized vapors 20 to drive formulation 18through spray-nozzle 14, thereby forming aerosol 24. The metered dose,contained in aerosol 24, is delivered via the inhaled air-stream to therespiratory tract of the subject via mouthpiece 22.

Another exemplary medical device for delivering an NO-donating compoundaccording to the present invention is a nebulizer inhaler. Nebulizerinhalers produce a stream of high velocity gas, typically from apressurized external source, which causes a therapeutic agent to sprayas a mist which is carried into the respiratory tract of a subject. Thetherapeutic agent is formulated in a liquid form, such as a solution ora suspension of micronized particles of respirable size, where asuspension of micronized particles is defined as being composed of atleast 90% of particles with a diameter of about 10 microns or less.Nebulizer inhalers are most adapted for use in a clinic or hospitalsetting. Nebulizer inhalers are advantageous for achieving substantiallycontinuous administration of therapeutic agent on a time-scale ofminutes, or longer. Typically a nebulizable therapeutic agent solutionis packaged in the form of single-dose vials, or in the form of amulti-dose bottle having a calibrated dropper.

Ample guidance for obtaining and utilizing a suitable nebulizer inhaleris provided in the literature of the art [refer, for example toO'Callaghan and Barry, 1997. Thorax 52(Suppl 2):S31-S44 S31].

An example of a nebulizer inhaler which is suitable for use as a medicaldevice according to the present invention (Venturi-type) is shown inFIG. 35. Nebulizer inhaler 26 includes: a nebulization chamber 28, afluid formulation 30, which comprises an NO-donating compound accordingto the present invention; a pressurized air delivery conduit 32,including pressurized air, an aerosol 34, an aerosol delivery conduit36, and an expiration conduit 38. For administration of the NO-donatingcompound via the nebulizer inhaler, pressurized air is delivered, viaair delivery conduit 32, to nebulization chamber 28. Upon exit fromnebulization chamber 28, the rapid flow of the exiting pressurized aircauses a negative pressure which sucks fluid formulation 30 upnebulization chamber 28 where it encounters exiting pressurized air andbecomes aerosol 34. Generated aerosol 34, containing the NO-donatingcompound, is delivered, via aerosol delivery conduit 36, to a subject,for example via a respiration mask. Upon expiration by the subject,nebulizer inhaler 26 continues to generate aerosol 34, but the expiredair and concomitantly generated aerosol 34 are wasted via expirationconduit 38 which allows one-way flow of the wasted gas from nebulizationchamber 28.

Another exemplary medical device for delivering an NO-donating compoundby inhalation, according to the present invention, is a vaporizer. Avaporizer consists of a hot air supply with temperature controls, avaporization chamber through which the hot air passes; a cooling chamberand an exhaust or mouthpiece. The compound or composition desired forvaporization, such as the NO-donating compound according to the presentinvention, is placed in the vaporization chamber, and the hot air bringsthe substance to the vapor phase by heat, and delivers it to the coolingchamber, typically a water reservoir through which the hot air and thevaporized substances are bubbled through and where the temperature isadjusted for the particular use, such as free release to an open spaceor direct inhalation mouthpiece.

Another exemplary medical device for delivering an NO-donating compoundby inhalation, according to the present invention, is an electricwarmer. An electric warmer typically consists of an electric heatingelement, and an open crucible, or other heat resistant container, inwhich volatile or volatilizable compositions or compounds are placed. Anelectric warmer according to this embodiment of the present inventiontherefore includes a volatile or volatilizable NO-donating compound ofthe present invention or a composition containing same, which is placed,directly or indirectly in the heat resistant container of the warmer.Another exemplary device for delivering an NO-donating compound byinhalation, according to the present invention, is a warmer, asdescribed above, in which the heating element is non-electric (e.g., acandle).

Exemplary respirators, according to an embodiment of this aspect of thepresent invention, can be breathing machines, body respirators (e.g.,cuirass), lung ventilators and resuscitators. These devices typicallyinclude a mask, a tube or a tent, connected to the respirator, throughwhich the active compound (the NO-donating compounds according to thepresent invention) is delivered. The active compound is introduced intothe airflow of the respirator by means of one of the techniquesdescribed hereinabove, e.g., by aerosolization via, for example anebulizer, or by evaporation via, for example, a vaporizer.

According to another embodiment of this aspect of the present invention,the medical device is such that delivering the NO-donating compound iseffected by topically applying the medical device on a biologicalsurface of a subject. The biological surface can be, for example, askin, scalp, an eye, an ear and a nail. Such medical devices can be usedin the treatment of various skin conditions and injuries, eye and earinfections and the like.

Exemplary medical devices for topical application include, withoutlimitation, an adhesive strip, a bandage, an adhesive plaster, a wounddressing and a skin patch.

Thus, in an embodiment of this aspect of the present invention, themedical device is a wound dressing, which can be utilized for topicalapplication of the NO-donors of the present invention. Wound dressingscontaining a therapeutically active agent are typically used fortreating cutaneous injuries.

Typically, a wound dressing medical device has a flat structureconsisting of an occlusive or semi-occlusive backing layer, non-adherentand absorbent layers, an active agent layer and a securing adhesive tapearound the perimeter of the device.

Exemplary commercial wound dressings include hydrocolloids such as,Comfeel®, Granuflex® and Tegasorb®. These dressings absorb water andswell to form a hydrogel. Hydrogels form a moist environment under thedressing and thus promotes wound healing. Hydrogel-based wound dressingstypically lose or absorb water depending on the state of hydration ofthe wound. Exemplary commercial hydrogels include Intrasite Gel®,Nu-Gel® Purilon Gel® and Sterigel®.

Other known wound dressings are based on foam. Exemplary commercialfoamable wound dressings include Lyofoam®, Allevyn®, Spyrosorb® andTielle®.

In another embodiment of this aspect of the present invention, themedical device is an adhesive plaster, which can be utilized for topicalapplication of the NO-donors of the present invention. Adhesive plasterscan be used for treating small and superficial cutaneous injuries as afirst-aid measure and/or longer treatment of minor injuries.

An adhesive plaster typically has a flat structure consisting of anocclusive or semi-occlusive backing layer and a non-adherent andpartially absorbent pad. The pad-side of the backing layer has adhesivemargins at both sides of the pad, or all around the pad. The pad isimpregnated or otherwise contains a layer of the active agent (herein,an NO-donating compound according to the present invention or apharmaceutical composition containing same).

Adhesive strips and bandages may also be used as medical devices fortopical delivery of one or more of the NO-donating compounds of thepresent invention.

In still another embodiment of this aspect of the present invention, themedical device is such that delivering the NO-donating compound iseffected transdermally. In this embodiment, the medical device isapplied on the skin of a subject, so as to transdermally deliver theNO-donating compound to the blood system.

Exemplary medical devices for transdermally delivering an NO-donoraccording to the present invention include, without limitation, anadhesive plaster and a skin patch. Medical devices for transdermal ortranscutaneous delivery of the NO-donating compound typically furtherinclude one or more penetration enhancers, for facilitating theirpenetration through the epidermis and into the system.

A schematic illustration of a typical skin patch, an exemplary medicaldevice according to this aspect of the present invention, which can beutilized for transdermal and/or topical delivery of the NO-donors of thepresent invention, is presented in FIG. 36. Thus, a skin patch 40comprises a backing 42, to which a reservoir 44 is attached. Reservoir44 contains a formulation 46 of the NO-donating compound according tothe present invention, optionally as a part of a pharmaceuticalcomposition. The reservoir can be, for example, a pad in which theNO-donating compound is dispersed or soaked, or a liquid reservoir. Thedevice can further include a frontal water or water vapor permeableadhesive 48, which adheres and secures the device to the treated region.Silicone rubbers with self-adhesiveness can alternatively be used. Inboth cases, a protective permeable layer 50 can be used to protect theadhesive side of the device prior to its use. Skin patch 40 may furthercomprise a removable cover 52, which serves for protecting the deviceupon storage.

In another embodiment of this aspect of the present invention, themedical device is such that delivering the NO-donating compound iseffected by implanting the medical device in a bodily organ. As usedherein, the term “organ” further encompasses a bodily cavity.

The organ can be, for example, a pulmonary cavity, a heart or heartcavity, a bodily cavity, an organ cavity, a blood vessel, an artery, avein, a muscle, a bone, a kidney, a capillary, the space between dermallayers, an organ of the female or male reproductive system, an organ ofthe digestive tract and any other visceral organ.

The medical device according to this embodiment of the presentinvention, typically includes a device structure in which an NO-donoraccording to the present invention is incorporated. The NO-donatingagents can thus be, for example, applied on, entrapped in or attached to(chemically, electrostatically or otherwise) the device structure.

The device structure can be, for example, metallic structure and thusmay be comprised of a biocompatible metal or mixture of metals (e.g.,gold, platinum).

Alternatively, the device structure may be comprised of otherbiocompatible matrices. These can include, for example, plastics,silicon, polymers, resins, and may include at least one component suchas, for example, polyurethane, cellulose ester, polyethylene glycol,polyvinyl acetate, dextran, gelatin, collagen, elastin, laminin,fibronectin, vitronectin, heparin, segmented polyurethane-urea/heparin,poly-L-lactic acid, fibrin, cellulose and amorphous or structured carbonsuch as in fullerenes, and any combination thereof.

In cases where a biodegradable implantable device is desired, the devicestructure can be comprised of a biocompatible matrix that isbiodegradable. Biodegradable matrices can include, for example,biodegradable polymers such as poly-L-lactic acid.

Optionally, the device structure may be comprised of biocompatiblemetal(s) coated with other biocompatible matrix.

Further optionally, in cases where a device which releases theNO-donating compounds of the present invention in a controlled manner isdesired, the device structure can be comprised of or coated with abiocompatible matrix that functions as or comprises a slow releasecarrier. The biocompatible matrix can therefore be a slow releasecarrier which is dissolved, melted or liquefied upon implantation in thedesired site or organ. Alternatively, the biocompatible matrix can be apre-determined porous material which entraps the NO-donating compoundsin the pores. When implanted in a desired site, the NO-donatingcompounds diffuse out of the pores, whereby the diffusion rate isdetermined by the pores size and chemical nature. Further alternatively,the biocompatible matrix can comprise a biodegradable matrix, which upondegradation releases the NO-donating compounds of the present invention.

The NO-donating compounds of the present invention can be incorporatedin the device structure by any methodology known in the art, dependingon the selected nature of the device structure. For example, theNO-donating compounds can be entrapped within a porous matrix, swelledor soaked within a matrix, or being adhered to a matrix.

Exemplary medical devices for delivering the NO-donating compounds ofthe resent invention by implanting include, without limitation, anaortic aneurysm graft device, an atrioventricular shunt, a catheter, adefibrilator, a heart valve, a hemodialysis catheter, a hemodialysisgraft, an indwelling arterial catheter, an indwelling venous catheter, aneedle, a pacemaker, a pacemaker lead, a patent foramen ovale septalclosure device, a stent, a stent graft, a suture, a synthetic vasculargraft, a thread, a tube, a vascular anastomosis clip, a vascularaneurysm occluder, a vascular clip, a vascular prosthetic filter, avascular sheath and a drug delivery port, a venous valve and a wire.

A preferred medical device according to this embodiment of the presentinvention is a stent. Stents, after insertion into blood vessels,oftentimes cause restenosis (the re-narrowing of a blood vessel). One ofthe most common complications after stent insertion is platelet adhesionand aggregation resulting in thrombus formation and lumen occlusion.Since NO is an antiplatelet agent and an anti-smooth muscle cellproliferation agent, it can consequently be used to reduce the risk ofthrombus formation associated with the use of stents, incorporatingNO-donors in stents is highly advantageous in this respect, especiallyif the bioactive residue conjugated to the NO-donor, according to thepresent invention, is an anti-inflammatory agent.

A schematic illustration of an exemplary implantable device according tothis embodiment of the present invention is presented in FIG. 37. FIG.37 presents stent 52, which is comprised of an expandable supportingelement 54 and further incorporates an active substance 56. The activesubstance can be either the NO-donating compounds according to thepresent invention per se, or, preferably, can be a biocompatible matrix,as described hereinabove, which comprises the NO-donating compounds ofthe present invention.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Chemical Syntheses

Materials and Methods:

Proton NMR spectra were recorded using Varian 300 MHz and Brucker 200MHz spectrometer. Spectra were obtained in deutro-chloroform (CDCl₃),unless indicated otherwise. Chemical shifts (δ) are reported in partsper million (ppm) downfield from tetramethylsilane.

Gas chromatography was carried out on a Hewlett-Packard (HP-6890) gaschromatograph having a 5% phenylsiloxan column and a FID detector.

Ultraviolet (UV) spectra were run as solutions in ethanol on a BeckmanMV1 spectrophotometer.

4-Methyl-5-thiazoleethanol and propionamide were purchased from Aldrichchemical Company, USA.

Phosphorus pentasulfide was purchased from Merck, Darmstadt, Germany.

Tetrahydrofuran (THF) was dried and was freshly distilled oversodium/benzophenone ketyl prior to use.

Other reagents were purchased from different suppliers and were usedwithout purification, unless otherwise indicated.

5-Acetoxy-3-chloro-2-pentanone (ACP) was prepared according to thesynthetic pathway described in Scheme 1 below and the correspondingprocedure published by Buchman [J. Am. Chem. Soc. 58, 1803, 1936].

Syntheses of 2-substituted-4-methyl-5-(2-nitrooxy-ethyl)-thiazoleDerivatives—

General Procedure:

The general synthetic pathway for preparing2-substituted-4-methyl-5-(2-nitrooxy-ethyl)-thiazole derivatives, whichserve as thiazole-based NO-donor compounds according to a preferredembodiment of the present invention, is presented in Schemes 2-4 below.In general, a desired thioamide is first prepared from a correspondingamide (Scheme 2), and is thereafter reacted, via a condensationreaction, with an alpha-chloroketone such as ACP, so as to form a4-substituted-5-thiazoleethanol derivative (a 4-methyl-5-thiazoleethanolderivative in case of ACP) (Scheme 3). The alcohol moiety of the latteris then reacted with nitric acid, so as to produce the desired NO-donoraccording to this preferred embodiment of the present invention (Scheme4).

Thus, according to a representative synthetic pathway, a desiredthioamide (general Compound II) is typically prepared according to thepresent invention by placing a corresponding amide (general Compound I)in a dry solvent such as THF or toluene, slowly adding theretophosphorus pentasulfide (P₂S₅), while stirring, for a time period of20-30 minutes under controlled temperature, and heating the resultingmixture at reflux temperature for additional 2-3 hours.

The respective 4-methyl-5-thiazoleethanol (general Compound III) isprepared from the thioamide (Compound II) according to the Hantzschprocedure [Hantzsch and Trauman, 1888, Ber 21, 938], as is shown inScheme 3 below, by adding to the thioamide reaction mixture5-acetoxy-3-chloro-2-pentanone (ACP), over a time period of 15-20minutes. The reaction mixture is then heated at reflux temperature forabout 20 hours, and the solvent is thereafter removed by distillation atatmospheric pressure. The reaction mixture is then cooled to 25° C.,hydrochloric acid (10%) is added, and the mixture is heated at refluxtemperature for one additional hour. Extraction of the reaction mixturewith dichloromethane, drying over sodium sulfate, and evaporation todryness, results in the desired 4-methyl-5-thiazoleethanol derivative.

Nitration of the 4-methyl-5-thiazoleethanol derivative (Compound III) isthen carried out as is illustrated in Scheme 4 below, by drop wiseaddition of nitric acid (85-90%) to sulfuric acid (95-98%) over a timeperiod of 20 minutes while keeping the temperature at 0-5° C., followedby addition of the 4-methyl-5-thiazoleethanol derivative. After stirringthe reaction mixture for additional 2-3 hours at 0-5° C., it is pouredcarefully onto cold water, washed with sodium hydroxide 20%, andextracted with dichloromethane. The extracts are combined, dried andevaporated to dryness, to thereby produce the respective2-substituted-4-methyl-5-(2-nitrooxy-ethyl)-thiazole (general CompoundIV, also denoted as Pet, whereby each specific derivative is identifiedby a numeral).

Using the general procedure described above, a variety of NO-donorcompounds according to an preferred embodiment of the present inventionwere prepared, as is detailed hereinbelow.

Preparation of 2-Ethyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-2)Synthesis of 2-(2-ethyl-4-methyl-thiazol-5-yl)-ethanol

Propionamide (20 grams, 0.27 moles) and dried THF (150 grams) were addedto a 500 ml three-neck flask equipped with a mechanical stirrer, acondenser and a thermometer. The mixture was stirred and 18 grams ofphosphorus pentasulfide (0.08 moles) were added over a time period of 30minutes while keeping the temperature between 20-25° C. The reactionmixture was then refluxed for 2 hours and was thereafter cooled to roomtemperature. 57.8 grams of 5-acetoxy-3-chloro-2-pentanone were thenadded, over a time period of 15 minutes, and the reaction mixture wasallowed to reflux for 20 hours. Thereafter, the THF was removed bydistillation at atmospheric pressure, the reaction mixture was cooled to25° C., 120 grams of hydrochloric acid (10%) were added, and the mixturewas brought back to reflux for 1 additional hour. After cooling to roomtemperature the reaction mixture was extracted with two portions of 50grams of dichloromethane to remove excess starting materials. The acidicaqueous phase was turned basic with a 20% aqueous solution of sodiumhydroxide and the resulting thiazole was extracted with three portionsof 75 grams of dichloromethane. The dichloromethane extracts werecombined and dried over sodium sulfate, filtered and evaporated todryness. The residue was distilled under vacuum at 0.5 mmHg to yield 20grams of pale yellow oil (43.3% yield) having a purity of 98.9%, asdetermined by gas chromatography.

¹H-NMR (CDCl₃): δ=3.75 (t, 2H, CH₂OH, J=6.6 Hz), 2.84 (t and qoverlapping, 5H, J=6.6 Hz and 7.5 Hz respectively), 2.26 (S, 3H, CH₃,benzylic), 1.29 (t, 3H, J=7.5 Hz) ppm;

UV (ethanol): λmax=252 nm (ε=56004).

MS: m/e (relative intensity)=171[M⁺ (27)], 140(100), 99(5), 85(12),45(20).

Synthesis of 2-Ethyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole

Fifteen (15) ml of nitric acid of (85-90%) were added dropwise to 15 mlof sulfuric acid (95-98%) over a time period of 20 minutes while keepingthe temperature at 0-5° C. After 2 hours, 14.3 grams (83.60 mmole) of2-ethyl-4-methyl-5-thiazoleethanol, prepared as described above, wereadded, while the temperature was still maintained at 0-5° C., thereaction mixture was stirred for 2 hours at the same temperature, andwas then poured carefully onto 125 ml of cold water. The water solutionwas made basic with a 20% aqueous solution of sodium hydroxide and theaqueous phase was extracted with three portions of 75 grams ofdichloromethane. The extracts were combined and dried over sodiumsulfate, filtered and evaporated to dryness. Chromatography of the crudeproduct on silica gel (using a mixture of 75:25 ethyl acetate:hexane asan eluent) yielded 10.5 grams (58% yield) of colorless liquid having apurity of 98%, as determined by gas chromatography.

¹H-NMR (CDCl₃): δ=1.23 (t, 3H, J=11.4 Hz), 2.22 (s, 3H, benzylic), 2.79(q, 2H, CH₂ benzylic, J=11.4 Hz), 3.05 (t, 2H, CH₂ benzylic, J=10.2 Hz),4.46 (t, 2H, CH₂ONO₂, J=10.2 Hz) ppm.

UV (ethanol): λmax=250 nm (ε=53070).

MS: m/e (relative intensity)=189(30), 140(100), 99(9), 85(10).

Preparation of 2,4-Dimethyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-3)

The respective ethanol derivative of Pet-3,2-(2,4-Dimethyl-thiazol-2-yl)-ethanol, was prepared according to thegeneral procedure described above for general Compound III and theprocedure described above for Pet-2, by reacting 10 grams (0.27 moles)of acetamide, 150 grams dried toluene and 18 grams (0.08 moles)phosphorus pentasulfide, and thereafter adding 48.20 grams of5-acetoxy-3-chloro-2-pentanone to the reaction mixture and refluxing theresulting mixture for 20 hours, after which the toluene was removed bydistillation at atmospheric pressure. The residue was then cooled to 25°C. and 120 grams of hydrochloric acid (10%) were added. The reactionmixture was allowed to reflux for 1 additional hour and, after cooling,was extracted with two portions of 50 grams of dichloromethane to removeexcess starting materials. The acidic water phase was turned basic with20% sodium hydroxide and the obtained thiazole was extracted with threeportions of 75 grams of dichloromethane. The dichloromethane extractswere combined and dried over sodium sulfate, filtered and evaporated todryness. The residue was distilled under vacuum at 2 mmHg to provide 20grams (43.3% yield) of colorless oil having a purity of 98.9% asdetermined by gas chromatography.

Nitration of 2-(2,4-Dimethyl-thiazol-2-yl)-ethanol, was carried out asdescribed above, to give 9.5 grams (48% yield) of2,4-dimethyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-3) as pale yellow liquidhaving a purity of 99.5% as determined by gas chromatography.

¹H-NMR (CDCl₃): δ=2.45 (s, 3H, CH₃), 2.72 (s, 3H, CH₃), 2.79 (q, 2H, CH₂benzylic, J=11.3 Hz), 3.05 (t, 2H, CH₂, J=10.1 Hz), 4.46 (t, 2H,CH₂ONO₂, J=10.2 Hz) ppm.

Preparation of 2-Isopropyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-4)

Based on the general procedure presented hereinabove, dry toluene (200ml) was placed in a 500 ml round-bottomed flask fitted with a refluxcondenser. A mixture of 30 grams (0.34 moles) finely grindedisobutyramide (prepared according to Kent R. E. and McElvain S. M.,Organic Syntheses, CV 3, 490) and 15.2 grams (0.07 moles) of powderedphosphorus pentasulfide was prepared and immediately transferred to theflask. In a separate vial a solution of 27 ml (0.34 moles) of5-acetoxy-3-chloro-2-pentanone (ACP) and 50 ml of dry toluene wasprepared, and 5 ml of this solution were added to the reaction mixture.The exothermic reaction was initiated by gradual heating in an oil bath,the oil bath was then removed and the remainder of the ACP-toluenesolution was added gradually through the reflux condenser. Uponcompleting the addition of ACP and once the reaction appeared to beceased, the mixture was heated at 75° C. for 24 hours. The reactionmixture was thereafter cooled to 25° C., 120 grams of hydrochloric acid(10%) were added and the mixture was refluxed for an additional onehour. After cooling to room temperature, the reaction mixture wasextracted with two portions of 50 grams of dichloromethane to removeexcess starting materials. 250 ml of water were added to the toluenephase while shaking for 30 minutes, the bi-phasic mixture was pouredinto a separation funnel, and the reddish organic layer was discarded.The aqueous layer was turned basic with 5 N sodium hydroxide orpotassium hydroxide, the crude thiazole, which separates as a blackorganic layer, was extracted with ether, and the aqueous lower layer waswashed with five 120 ml portions of ethyl acetate. The combined ethylacetate extracts were dried over anhydrous sodium sulfate and filteredthrough glass wool. The ethyl acetate was removed by steam bathdistillation, to give 21 grams (21% yield based on isobutyramide) ofresidual oil having a purity of 98% as determined by gas chromatography.

¹H-NMR (CDCl₃): δ=2.45 (s, 3H), 2.22 (s, 3H, benzylic), 2.79 (q, 2H, CH₂benzylic, J=11.4 Hz), 3.05 (t, 2H, CH₂ benzylic, J=10.2 Hz), 4.46 (t,2H, CH₂ONO₂, J=10.2 Hz) ppm.

UV (ethanol): λmax=250 nm (ε=53070).

MS: m/e (relative intensity)=189(30), 140(100), 99(9), 85(10).

Nitration of 2-(2-isopropyl-4-methyl-thiazol-2-yl)-ethanol, was carriedout as described above, to give 1.5 grams (50% yield) of2-isopropyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-4) having apurity of 98% as determined by gas chromatography.

Preparation of4-Methyl-5-(-2-nitrooxy-ethyl)-2-(1-nitrooxy-ethyl-thiazole (Pet-5)

Pet-5 was prepared according to the general procedure presentedhereinabove and the procedure described above for the preparation ofPet-2, using L-(−)-lactamide as the starting material and dry THF as asolvent, to give the product as brown oil (56% yield) having a purity of98% as determined by thin-layer chromatography.

¹H-NMR (CDCl₃): δ=1.51 (s, 3H, CH₃), 2.49 (s, 3H, CH₃, Aromatic), 2.79(t, 2H, CH₂ benzylic), 3.85 (t, 2H, CH₂), 4.69 (q, 1H, CHONO₂) ppm.

Preparation of 4-Methyl-5-(nitrooxy-ethyl)-2-trifluoromethyl-thiazole(Pet-6)

Pet-6 was prepared according to general procedure presented hereinaboveand the procedure described above for the preparation of Pet-2, usingtrifluoroacetamide as the starting material and dry toluene as asolvent, to give the product as pale yellow oil (35% yield) having apurity of 96% as determined by thin-layer chromatography.

¹H-NMR (CDCl₃): δ=2.45 (s, 3H, CH₃), 2.74 (q, 2H, CH₂, J=11.2 Hz), 3.10(t, 2H, CH₂, J=10.2 Hz), 3.89 (t, 2H, CH₂ONO₂, J=10.3 Hz) ppm.

Preparation ofdimethyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine (Pet-7)

The synthesis of Pet-7 is illustrated in Scheme 5 below.

According to the general procedure presented hereinabove, 200 ml of drytoluene and 10 grams (0.095 moles) of N,N-dimethylthiourea (obtainedfrom Merck, Germany) were placed in a 500 ml round-bottomed flask fittedwith a reflux condenser. 17.5 grams (0.1 moles) of5-acetoxy-3-chloro-2-pentanone (ACP) were added to the solution over atime period of 20 minutes. The reaction mixture was refluxed at 80° C.for 24 hours, after which the toluene was removed by evaporation. 100 mlof water and 15 ml of HCl solution (32%) were added and the resultingmixture was refluxed for 1 hour at 90° C. After cooling, the mixture waswashed with two portions of 100 ml of dichloromethane to remove excessof starting materials. The aqueous phase was turned basic (pH 8-9) using5 N solution of sodium hydroxide. The2-(2-dimethylamino-4-methyl-thiazol-5-yl)-ethanol was extracted withthree portions of 100 ml of dichloromethane and the combined extractswere dried over sodium sulfate. The dichloromethane was removed undervacuum to yield 8 grams (45%) of white crystalline powder, which wasused in the subsequent nitration step without further purification.

Nitration of 2-(2-dimethylamino-4-methyl-thiazol-5-yl)-ethanol wascarried out by cooling 1.58 grams (0.016 moles) of sulfuric acid(95-98%) to 0-5° C. and addition thereto of 1.016 grams (0.016 moles)nitric acid (70%) drop-wise over a time period of 20 minutes whilekeeping the temperature between 0-5° C. Following, 3 grams of2-(2-dimethylamino-4-methyl-thiazol-5-yl)-ethanol (16.10 mmoles) wereadded over a time period of 30 minutes at 0-5° C. After the addition wascompleted, the reaction mixture was stirred for 30 minutes at 0-5° C.The reaction mixture was then added carefully to 25 ml of cold water.The water solution was turned basic with a 20 5 aqueous solution ofsodium hydroxide and the aqueous phase was extracted with three 75 gramsportions of a mixture of 1:1 ethyl acetate:ether. The organic extractswere combined, dried over sodium sulfate, filtered and evaporated todryness. The nitration step was confirmed initially by thin-layerchromatography stained with diphenylamine as a marker for the nitrateester moiety (FIG. 1). Chromatography of the crude product on silica gel(using a mixture of 2:1 ethyl acetate:hexane as eluent) gave 2 grams(54% yield) of the product as white crystalline needles having a purityof 99% as determined by gas chromatography.

¹H-NMR (CDCl₃): δ=2.45 (s, 6H, N—CH₃), 2.54 (s, 3H, CH₃), 2.75 (q, 2H,CH₂), 3.12 (t, 2H, CH₂), 4.01 (t, 2H, CH₂ONO₂) ppm.

Preparation of 4-methyl-5-(2-nitrooxy-ethyl)-2-phenyl-thiazole (Pet-8)

2-(4-Methyl-2-phenyl-thiazol-5-yl)-ethanol was prepared, according togeneral procedures presented hereinabove, by adding 7.3 grams (0.053moles) of thiobenzamide (obtained from Merck, Germany) to 200 ml dryTHF, followed by addition of 9.5 grams (0.053 moles)5-acetoxy-3-chloro-2-pentanone over a time period of 20 minutes. Thereaction mixture was then refluxed at 80° C. for 24 hours, after whichthe THF was removed by evaporation. 100 ml of water and 15 ml of HClsolution (32%) were added and the reaction mixture was refluxed for 1hour at 90° C. After cooling, the mixture was washed with two portionsof 100 ml of dichloromethane to remove excess starting materials. Theaqueous phase was turned basic (pH 8-9) using an aqueous solution of 5 Nsodium hydroxide. The 2-(4-methyl-2-phenyl-thiazol-5-yl)-ethanol wasextracted with three portions of 100 ml of dichloromethane and thecombined extracts were dried over sodium sulfate. After removal of thedichloromethane 8 grams (45% yield) of the2-(4-methyl-2-phenyl-thiazol-5-yl)-ethanol product were obtained as areddish-brown liquid.

The nitration of 2-(4-methyl-2-phenyl-thiazol-5-yl)-ethanol was carriedout subsequently according to the procedure described hereinabove, togive 4-methyl-S-(2-nitrooxy-ethyl)-2-phenyl-thiazole) in 40% yield. Theproduct was purified by column chromatography using a mixture of 1:1:1ethyl acetate:hexane:dichloromethane as eluent, to purity of 99.5% asdetermined by HPLC and by thin-layer chromatography stained withdiphenylamine as a marker for the nitrate ester moiety (FIG. 1).

NMR (CDCl₃): δ=2.37 (s, 3H, CH₃), 3.18 (t, 3H, CH₂), 4.57 (t, 2H,CH₂ONO₂), 7.32-8.19 (m, 5H, Aromatic) ppm.

Preparation of 2-methoxy-4-methyl-5-(2-nitrooxy-ethyl)-thiazole (Pet-9)

Pet-9 was prepared according to general procedure presented hereinaboveand the procedure described above for the preparation of Pet-2, usingthe respective amide as the starting material and dry THF as a solvent,to give the product as brown oil (51% yield) having a purity of 97% asdetermined by thin-layer chromatography.

The product was further purified by column chromatography using amixture of 1:1 ethyl acetate:hexane as eluent, to purity of 99.5% asdetermined by gas chromatography.

¹H-NMR (CDCl₃): δ=2.45 (s, 3H, CH₃, benzylic), 3.72 (s, 3H, OCH₃), 4.46(t, 2H, CH₂ONO₂, J=10.2 Hz) ppm.

Preparation of 4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-ylamine (Pet-10)

2-(2-Amino-4-methyl-thiazol-5-yl)-ethanol was prepared according togeneral procedures presented hereinabove, by adding 20 grams (0.263moles) of thiourea to 200 ml of dry toluene, followed by addition of 47grams (0.263 moles) of 5-acetoxy-3-chloro-2-pentanone over a time periodof 20 minutes. The reaction mixture was heated at 80° C. for 24 hoursand thereafter approximately 180 ml of toluene were removed byevaporation. 180 ml of water and 20 ml of HCL solution (32%) were thenadded and the reaction mixture was refluxed for 1 hour at 90° C. Theorganic phase was then removed by washing with chloroform, and theaqueous phase was turned basic (pH 8-9) using a 5 N solution of sodiumhydroxide. The product was extracted once with 70 ml chloroform and oncewith 70 ml ethyl acetate. The combined organic extracts were dried oversodium sulfate and the solvents were removed by evaporation to give 30grams (72% yield) of violet-brown crystals.

Subsequently, 4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-ylamine wasprepared by drop-wise addition of 1.59 grams of nitric acid (70%) to2.48 grams of cooled sulfuric acid (95-98%) at 0-5° C. over a timeperiod of 20 minutes. Following, 4 grams (0.025 moles) of2-(2-Amino-4-methyl-thiazol-5-yl)-ethanol were added over a time periodof 45 minutes at 0-5° C. After the addition was completed, the reactionmixture was stirred for 3 hours at 25° C., and was then added carefullyto 50 ml of cold water. The water solution was turned basic with anaqueous solution of 20% sodium hydroxide and the aqueous phase wasextracted with three portions of 75 grams of ethyl acetate. The organicextracts were combined, dried over sodium sulfate, filtered andevaporated to dryness under vacuum. Chromatography of the crude producton silica gel (using a mixture of 8:2 ethyl acetate:hexane as eluent)gave 1.8 grams (35% yield) of the product as orange oil having a purityof 98.5% as determined by gas chromatography.

¹H-NMR (CDCl₃): δ=2.10 (s, 3H, CH₃), 2.79 (t, 2H, CH₂), 3.37 (t, 2H,CH₂OH), 4.87 (s, breit, NH₂) ppm.

Preparation of 4-[4-methyl-5-(2-nitrooxy-ethyl)thiazole-2-yl]-piperidine(Pet-11)

Pet-11 was prepared according to general procedure presented hereinaboveand the procedure described above for the preparation of Pet-2, usingthe respective amide as the starting material and dry THF as a solvent,to give the product as red oil (56% yield) having a purity of 95% asdetermined by thin-layer chromatography.

¹H-NMR (CDCl₃): δ=1.77 (t, 2H, CH₂,-piperidine), 2.45 (t, CH₃-benzylic),2.77 (t, 2H, CH₂,-piperidine), 2.73 (t, 2H, CH₂-benzylic) 2.78 (t, H,CH,-piperidine), 4.45 (t, 2H, CH₂—ONO₂) ppm.

Preparation of 3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-yl]-pyridine(Pet-12)

2-(4-Methyl-2-pyridin-3-yl-thiazole-5-yl)-ethanol was prepared accordingto the general procedure presented hereinabove, by adding 20 grams(0.145 moles) of thionicotinamide (purchased from Acros, Belgium) to 200ml of dry toluene, followed by addition of 26 grams (0.145 moles) of ACPover a time period of 20 minutes. The reaction mixture heated for 24hours at 80° C. and thereafter about 180 ml of toluene were removed byevaporation. 100 ml of water and 20 ml of HCl solution (32%) were addedand reflux was continued for 1 hour at 90° C. The organic phase was thenremoved by washing with chloroform and the aqueous phase was turnedbasic (pH 8-9) using a 5 N solution of NaOH. The2-(4-methyl-2-pyridin-3-yl-thiazole-5-yl)-ethanol was extracted withthree portions of 100 ml of chloroform and the combined extracts weredried over sodium sulfate. The chloroform was thereafter removed and theresidue was purified by liquid chromatography, using a mixture of 9:1ethyl acetate:methanol as eluent, to give 10 grams (31% yield) of the2-(4-methyl-2-pyridin-3-yl-thiazole-5-yl)-ethanol product as aviolet-brown powder.

3-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-yl]-pyridine was prepared bydrop-wise addition of 1.145 gr. of nitric acid (70%) to 1.78 grams ofcooled sulfuric acid (95-98%) at 0-5° C., followed by addition of 4grams (0.0181 moles) 2-(4-methyl-2-pyridin-3-yl-thiazole-5-yl)-ethanolover a time period of 30 minutes at 0-5° C. After the addition wascompleted the reaction mixture was stirred for 30 minutes at 0-5° C.,and for one additional hour at room temperature. The reaction mixturewas then added carefully to 25 ml of cold water. The water solution wasturned basic with an aqueous 20% solution of sodium hydroxide and theaqueous phase was extracted with three portions of 75 grams ofdichloromethane. The extracts were combined, dried over sodium sulfate,filtered and evaporated to dryness under vacuum. The nitration step wasconfirmed initially by thin-layer chromatography stained withdiphenylamine as a marker for the nitrate ester moiety (FIG. 1).Chromatography of the crude product on silica gel (using ethyl acetateas eluent) gave 1.5 grams (41% yield) of Pet-12 as a pale yellow liquidhaving a purity of 99% as determined by HPLC.

¹H-NMR (CDCl₃): δ=2.47 (s, 3H, CH₃), 2.77 (t, 2H, CH₂, benzylic), 3.88(t, 2H, CH₂ONO₂), 7.44-8.56 (m, 4H, Aromatic) ppm.

Preparation of1,4-Bis-[4-Methyl-5-(2-nitrooxy)-ethyl)-thiazol-2-yl]-Butane (Pet-13)

Pet-13 was prepared according to general procedure presented hereinaboveand the procedure described above for the preparation of Pet-2, usingthe respective hexane diamide as the starting material and dry THF as asolvent, to give the product as red oil (52% yield) having a purity of98% as determined by thin-layer chromatography stained withdiphenylamine as a marker for the nitrate ester moiety (FIG. 1).

Preparation of2-[1-(6-methoxy-naphthalen-2-yl-ethyl]-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-17)

The starting material naproxenamide was prepared as described in Scheme6 below.

Naproxyl chloride was first prepared in a 500 ml three-necked flaskequipped with a 250 ml dropping funnel, an efficient mechanical stirrer,and a condenser with a gas-absorption trap attached to the top of thecondenser. The water supplied to the condenser was cooled to 0° C., andthe flask was cooled in a large water bath. 15.5 grams (0.13 moles) ofthionyl chloride were placed in the flask, and 30 grams (0.13 moles) ofnaproxen were added while rapidly stirring the reaction mixture (avigorous evolution of hydrogen chloride and sulfur dioxide wasobserved). After completing the acid addition, the water bath was heatedat 80° C. for 30 minutes while stirring.

Naproxenamide was then prepared in a 1-liter flask surrounded by anice-salt freezing mixture and equipped with an efficient mechanicalstirrer and a 500 ml dropping funnel. 125 ml of cold concentratedaqueous ammonia (about 25%) were placed in the flask and 20 grams (0.08moles) naproxyl chloride were added drop-wise while rapidly stirring thereaction mixture and maintaining the temperature below 15° C., and theevolution of white fumes (mostly ammonium chloride) at a controllablerate. Stirring was continued for one hour after the addition of the acidchloride was completed and thereafter the reaction mixture wasevaporated to dryness under vacuum. The dry residue of ammonium chlorideand naproxenamide was boiled for 10 minutes with 300 ml of dry ethylacetate, and the boiling solution was filtered rapidly through a flutedfilter paper on a large hot funnel. The residue on the filter wasextracted with two portions of 100 ml of ethyl acetate, the combinedethyl acetate extracts were cooled to 0° C., and the precipitatedcrystalline amide was removed by filtration. The filtrate wasconcentrated to about 30 ml, chilled, and a second crop of amide wascollected. The two crops of naproxenamide were combined and dried, firstin an oven at 70° C. for 3 hours and then in a vacuum desiccator, toyield 15 grams (80% yield) of naproxen amide as glistening whiteneedles.

Pet-17 was then prepared from naproxenamide according to the generalprocedure described above and the procedure for the preparation ofPet-2, as is illustrated in Scheme 7 below. Pet-17 was obtained in anoverall yield of 25%.

¹H-NMR (CDCl₃): δ=1.72 (s, 3H, CH₃), 2.42 (s, 3H, CH₃), 2.81 (t, 2H,CH₂), 3.75 (s, 3H, OCH₃), 3.85 (t, 2H, CH₂ONO₂), 4.33 (q, 1H, CH),7.11-7.58 (m, 6H, aromatic) ppm.

Preparation of4-methyl-5-(2-nitrooxy-ethyl)-2-(4-trifluoromethyl-phenyl)-thiazole(Pet-59)

Pet-59 was prepared according to general procedure presented hereinaboveand the procedure described above for the preparation of Pet-7, using4-(trifluoromethyl)-thiobenzamide (obtained from Avocado, UK) as thestarting material. The respective alcohol was obtained in 73% yield asbrown crystals whereby Pet-59 was obtained as pale brown crystals (74%yield) having a purity of 99% as determined by thin-layer chromatographyand gas chromatography.

¹H-NMR (CDCl₃) of the alcohol intermediate: δ=2.42 (s, 3H, CH₃), 3.01(t, 2H, CH₂, J=6 Hz), 3.85 (t, 2H, CH₂OH, J=6 Hz) ppm.

¹H-NMR of Pet-59 (CDCl₃): δ=2.43 (s, 3H, CH₃), 3.18 (t, 2H, CH₂, J=6.6Hz), 4.60 (t, 2H, CH₂ONO₂, J=6.6 Hz) ppm.

Preparation of2-[1-(4-isobutyl-phenyl)-ethyl]-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-66)

Pet-66 was prepared according to general procedure presented hereinaboveand the procedure described above for the preparation of Pet-2, usingthe respective amide as the starting material, in an overall 35% yield.

¹H-NMR (CDCl₃): δ=2.68 (s, 3H, CH₃), 2.82 (q, 2H, CH₂ benzylic, J=11.3Hz), 3.86 (t, 2H, CH₂ONO₂, J=10.2 Hz) ppm.

Preparation of Bis-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-diazene(Pet-102)

Pet-102 was prepared according to general procedure presentedhereinabove and the procedure described above for the preparation ofPet-3, using azodicarbonamide (Aldrich-Sigma) as the starting material,in an overall yield of 55%.

¹H-NMR (CDCl₃): δ=2.62 (s, 6H, 2×CH₃), 2.82 (q, 4H, 2×CH₂), 3.86 (t, 4H,2×CH₂ONO₂) ppm.

Preparation of acetic acid2-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl ester (Pet-116)

The synthesis of Pet-116 is illustrated in Scheme 8 below.

2-[5-(2-Hydroxy-ethyl)-4-methyl-thiazol-2-yl)-phenol was prepared fromthe respective amide, according to the general procedure describedabove, using toluene as the solvent, to give a pink powder (55% yield),which was purified by column chromatography on silica gel using amixture of 3:2:2 ethyl acetat:chloroform:hexane as eluent.

Nitration of 2-[5-(2-Hydroxy-ethyl)-4-methyl-thiazol-2-yl)-phenol wascarried out according to the procedures described above, to give2-[4-Methyl-5-(2-nitrooxy-ethyl-thiazol-2-yl]-phenol as a yellow powder(63% yield).

2-[4-Methyl-5-(2-nitrooxy-ethyl-thiazol-2-yl]-phenol was then subjectedto an acetylation reaction, which was carried out according to theprocedure described in Organic Syntheses, CV 3, 452, to give the finalproduct in 70% yield.

¹H-NMR (CDCl₃): δ=2.10 (s, 3H, COCH₃), 2.42 (s, CH₃, Aromatic), 3.75 (t,2H, CH₂-benzylic), 3.87 (t, CH₂ONO₂), 7.13-7.47 (m, 4H, Aromatic) ppm.

Preparation of4,4′-dimethyl-5,5′-bis-(2-nitrooxy-ethyl)-[2,2′]bitiliazolyl (Pet-118)

Pet-118 was prepared according to the general procedure presentedhereinabove, using dithiooxalamide (obtained from Merck, Germany) as astarting material. The synthesis of the respective alcohol,2-[5′-(2-Hydroxy-ethyl)-4,4′-dimethyl-[2,2′]bithiazolyl-5-yl]-ethanol isillustrated in Scheme 9 below.

Pet-118 was obtained in 43% yield, as a yellow powder having a purity of97%, as determined by TLC.

¹H-NMR (CDCl₃): δ=2.45 (s, 3H, CH₃), 2.79 (q, 2H, CH₂ benzylic, J=11.1Hz), 3.05 (t, 2H, CH₂, J=10 Hz), 4.46 (t, 2H, CH₂ONO₂, J=10.2 Hz) ppm.

Using the general procedure and the exemplary procedures describedabove, Pet-2, Pet-3, Pet-4, Pet-5, Pet-6, Pet-7, Pet-9, Pet-11, Pet-13,Pet-17, Pet-44, Pet-55, Pet-66, Pet-97, Pet-116, Pet-118, Pet-181,Pet-182, Pet-183, Pet-184, Pet-185 and Pet-186 presented in Tables 1 and2, were prepared and analyzed.

Other 2-substituted-4-methyl-5-(2-nitrooxy-ethyl)-thiazoles, aspresented, for example, in Tables 1 and 2, are similarly prepared.

Preparation of NO-Donors Having a Biocleavable Moiety—General Procedure:

The procedure presented hereinbelow is general procedure for thepreparation of thiazole-based NO-donor according to a preferredembodiment of the present invention, having a biocleavable moietybetween the thiazole residue and an additional moiety that is linedthereto. This procedure relies on the general synthetic pathway forpreparing the desired 2-(2-substituted-4-methyl-thiazol-5-yl)-ethanolderivative (Compound III) as presented hereinabove and described inSchemes 2 and 3, which serve as thiazole-based NO-donor compoundsaccording to a preferred embodiment of the present invention.

The general procedure is presented in Scheme 10 below. In general, areactive derivative of a 2-(2-substituted-4-methyl-thiazol-5-yl)-ethanol(Compound V, Scheme 10), is first prepared, and is thereafter reactedwith a desired compound having a second reactive group (K—X, Scheme 10).The first and the second reactive groups are selected capable ofreacting therebetween, to thereby form a biocleavable moiety (A, Scheme10). The resulting compound (Compound VI) thus includes a thiazolemoiety and a residue of the desired compound covalently linkedtherebetween by a biocleavable moiety.

Thus, according to a representative synthetic pathway, the firstreactive group on the thiazole derivative (Q in Compound V, Scheme 10)is, for example, an amine, the corresponding second reactive group onthe desired compound (K) is, for example, a carboxylic acid, and theformed biocleavable moiety is, for example, an amide. The synthesis inthis case is effected by adding dicyclohexylcarbodiimide (DCC) to anequal molar amount of the carboxylic acid derivative in dichloromethane.The mixture is stirred for 2 hours, followed by the addition of an equalmolar amount of 4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-ylamine(Pet-10, prepared as described hereinabove). The reaction mixture isstirred for 8 hours, after which the organic layer is removed and washedwith 5% NaOH solution followed by 5% HCl solution and finally with twoportions of water to remove excess starting materials. Thedichloromethane is dried using sodium sulfate and removed by evaporationto afford the corresponding[5-(2-hydroxy-ethyl)-4-methyl-thiazol-2-yl]-amide.

In accordance with the representative general synthetic pathwaypresented hereinabove, the nitration of the[5-(2-hydroxy-ethyl)-4-methyl-thiazol-2-yl]-amide is afforded by theaddition of 70% nitric acid to acetic anhydride while stirring andmaintaining the temperature between 20-30° C. by external cooling. Themixture is then cooled to −5° C. while stirring, followed by theaddition of the [5-(2-hydroxy-ethyl)-4-methyl-thiazol-2-yl]-amide. Themixture is kept for 30 minutes at −5° C. and then heated to 10° C. andstirred for one hour. The resulting mixture is poured thereafter intoice water and stirred for 1 hour. Aliquots of NaHCO₃ are added until CO₂evolution ceases. The aqueous phase is extracted with three portions ofethyl acetate, and the combined extracts are dried over sodium sulfateand concentrated by evaporation (Scheme 11).

In a second example, the first reactive group on the thiazole derivative(Q in Compound V, Scheme 10) is, for example, hydroxyl, thecorresponding second reactive group on the desired compound (K) is, forexample, a carboxylic acid, and the formed biocleavable moiety is, forexample, an ester. The synthesis in this case is executed by reacting anequal molar amount of the carboxylic acid derivative and an equal molaramount of the thiazole derivative in the presence of a catalytic amountof an acid or an equal molar amount of a base.

In another example, the first reactive group on the thiazole derivative(Q in Compound V, Scheme 10) is, for example, a carboxylic acid, and thecorresponding second reactive group on the desired compound (K) is, forexample, hydrazine, and the formed biocleavable moiety is, for example,hydrazide. The synthesis in this case is effected by an equal molaramount of the carboxylic acid derivative and an equal molar amount ofthe thiazole derivative in the presence of a catalytic amount of anacid.

The nitration step for the general examples above is carried out inaccordance with the representative general synthetic pathway for thebiocleavable amide described hereinabove.

Using the general procedure described hereinabove, a variety of NO-donorcompounds having a biocleavable moiety according to the presentinvention were prepared, as is detailed hereinbelow.

Preparation ofN-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-nicotinamide (Pet-154)

According to the general procedure presented hereinabove, 16.7 grams(0.081 mol) of dicyclohexylcarbodiimide (DCC) were added to 10 grams(0.081 mol) of nicotinic acid in dichloromethane. The mixture wasstirred for 2 hour, followed by the addition of 12.8 grams (0.081 mol)of 2-(2-amino-4-methyl-thiazol-5-yl)-ethanol. The reaction mixture wasstirred for additional 8 hours, after which the organic layer wasremoved and washed with 5% NaOH solution followed by 5% HCl solution andfinally with two portions of water to remove excess starting materials.The dichloromethane was dried using sodium sulfate and removed byevaporation to afford 13 grams (61%) of pale yellow powder ofN-[5-(2-hydroxy-ethyl)-4-methyl-thiazol-2-yl]-nicotinamide, as describedin Scheme 12 hereinbelow.

70% nitric acid (1.2 ml) was added to acetic anhydride (5 ml) whilestirring and maintaining the temperature between 20-30° C. by externalcooling. The mixture was cooled to −5° C. while stirring, followed bythe addition of 1 gram ofN-[5-(2-hydroxy-ethyl)-4-methyl-thiazol-2-yl]-nicotinamide. After 30minutes at −5° C. the mixture was heated to 10° C. and stirred for oneadditional hour. The resulting mixture was poured into ice-water andstirred for 1 hour. Aliquots of NaHCO₃ were added until CO₂ evolutionceased. The orange-yellow water solution was extracted with threeportions of 15 ml ethyl acetate. The combined extracts were dried oversodium sulfate and concentrated by evaporation.

¹H-NMR (CDCl₃): δ=2.48 (s, 3H, CH₃), 3.39 (q, 2H, CH₂ benzylic), 4.08(t, 2H, CH₂ONO₂), 7.24-8.87 (m, 4H, Pyridinic) ppm.

Preparation of2-(6-methoxy-naphthalen-2-yl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propionamide(Pet-150)

Pet-150 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using 2-(5-methoxy-naphthalen-2-yl)-propionic acid (Naproxen)and 4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as thestarting materials, in an overall yield of 43%.

¹H-NMR (CDCl₃): δ=1.56 (d, 1H, CH-naphtylic), 2.37 (s, 3H, CH₃), 2.49(s, 3H, CH₃), 3.33 (q, 2H, CH₂ benzylic), 3.89 (s, 3H, O—CH₃) 4.01 (t,2H, CH₂ONO₂), 7.04-7.67 (m, 4H, naphthylic) ppm.

Preparation of 5-[1,2]Dithiolan-3-yl-pentanoic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amide (Pet-151)

Pet-151 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using 5-[1,2]Dithiolan-3-yl-pentanoic acid (DL-Lipoic acid) and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 51% as a pale yellow liquid.

¹H-NMR (CDCl₃): δ=1.61 (m, 2H, CH₂, alpha-S), 1.66 (m, 2H, CH₂, beta-S),1.90 (m, 2H, CH₂, beta-C═(O)N), 2.25 (s, 3H, CH₃), 2.36 (m, 2H, CH₂),2.89 (t, 2H, CH₂, 3.54 (m, 1H, CH—S—CH₂), 3.80 (t, 2H, CH₂—ONO₂), 6.91(s, 1H, NH-amide) ppm.

Preparation of2-(4-Isobutyl-phenyl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propionamide(Pet-152)

Pet-152 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using 2-(4-isobutyl-phenyl)-propionic acid (Ibuprofen) and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 63%.

¹H-NMR (CDCl₃): δ=0.86 (m, 6H, CH—(CH ₃)₂, 1.18 (t, 2H, CH₂), 1.57 (d,1H, CH—CH₃), 2.21 (s, 3H, CH₃), 2.70 (m, 2H, CH₂), 3.06 (t, 2H, CH₂),3.41 (q, 3H, CH₃—CH,), 3.81 (q, 1H, CH₃—CH), 4.52 (t, 2H, CH₂—ONO₂),7.12-7.83 (m, 4H, aromatic) ppm.

Preparation ofN-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-1-oxy-nicotinamide(Pet-156)

Pet-156 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using 1-oxy-nicotinic acid and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 71%.

¹H-NMR (CDCl₃): δ=2.49 (s, 3H, CH₃), 3.32 (q, 2H, CH₂ benzylic), 4.18(t, 2H, CH₂ONO₂), 7.26-8.98 (m, 4H, Pyridinic) ppm

Preparation of4-acetylamino-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-benzamide(Pet-157)

Pet-157 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using 4-acetylamino-benzoic acid and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 44%.

¹H-NMR (CDCl₃): δ=2.48 (s, 3H, CH₃), 3.29 (q, 2H, CH₂ benzylic), 3.90(s, 3H, CH₃), 4.01 (t, 2H, CH₂ONO₂), 6.66-7.87 (m, 4H, aromatic) ppm

Preparation of hexadecanoic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amide (Pet-158)

Pet-158 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using hexadecanoic acid (palmitic acid) and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 62%.

Preparation of acetic acid2-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylcarbamoyl]-phenyl ester(Pet-159)

Pet-159 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using 2-acetoxy-benzoic acid (Aspirin) and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 25%.

¹H-NMR (CDCl₃): δ=2.44 (s, 3H, CH₃), 3.33 (q, 2H, CH₂ benzylic), 3.85(s, 3H, CH₃), 4.01 (t, 2H, CH₂ONO₂), 7.02-7.87 (m, 4H, aromatic) ppm

Preparation of Pyrrolidine-2-carboxylic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amide (Pet-160)

Pet-160 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using pyrrolidine-2-carboxylic acid and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 55%.

¹H-NMR (CDCl₃): δ=1.60 (m, 2H, CH₂, Pyrolidine), 1.88 (m, 2H, CH₂,pyrolodine, 1-beta-C(═O)—N), 2.44 (s, 3H, CH₃), 2.78 (m, 2H, CH₂,pyrolodine), 3.38 (q, 2H, CH₂ benzylic), 3.88 (s, 3H, CH₃), 4.07 (t, 2H,CH₂ONO₂) ppm.

Preparation of2,6-difluoro-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-benzamide(Pet-161)

Pet-161 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using 2,6-difluoro-benzoic acid and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 72%.

¹H-NMR (CDCl₃): δ=2.46 (s, 3H, CH₃), 3.34 (q, 2H, CH₂ benzylic), 4.12(t, 2H, CH₂ONO₂), 7.24-8.88 (m, 3H, aromatic) ppm.

Preparation of2-(2,4-dichloro-phenyl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide(Pet-162)

Pet-162 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using (2,4-dichloro-phenyl)-acetic acid and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 69%.

¹H-NMR (CDCl₃): δ=2.47 (s, 3H, CH₃), 3.38 (q, 2H, CH₂ benzylic), 4.12(t, 2H, CH₂ONO₂), 7.24 (dd, 2H, aromatic), 8.45 (s, 1H, aromatic) ppm.

Preparation of2-(2,4-dichloro-phenoxy)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide(Pet-163)

Pet-163 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using (2,4-dichloro-phenoxy)-acetic acid and4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylamine (Pet-10) as the startingmaterials, in an overall yield of 77%.

¹H-NMR (CDCl₃): δ=2.36 (s, 3H, CH₃), 3.30 (q, 2H, CH₂ benzylic), 4.05(t, 2H, CH₂ONO₂), 4.45 (s, 2H, CH₂), 7.11 (dd, 2H, aromatic), 8.23 (s,1H, aromatic) ppm.

Preparation of 4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acidN′-phthalazin-1-yl-hydrazide (Pet-153)

Pet-153 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-154, using phthalazin-1-yl-hydrazine and5-(2-Hydroxyethyl)-4-methylthiazole-2-carboxylic acid as the startingmaterials, in an overall yield of 47%.

¹H-NMR (CDCl₃): δ=2.39 (s, 3H, CH₃), 3.33 (t, 2H, CH₂ benzylic), 3.85(t, 2H, CH₂ONO₂), 4.45 (s, 1H, NH aromatic C—NH), 7.66-8.87 (m, 5H,aromatic) ppm.

Preparation of allyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine(Pet-155)

Pet-155 was prepared according to the general procedure describedhereinabove and the procedure described above for the preparation ofPet-7, using allylthiourea and ACP as the starting materials, in anoverall yield of 87%.

Preparation of 4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acid10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-ylester (Pet-164)

Pet-164 was prepared by a catalytic acid esterification reaction of5-(2-Hydroxyethyl)-4-methylthiazole-2-carboxylic acid with commercialavailable (Sigma) testosterone to afford the title compound in anoverall yield of 34%.

Other 2-substituted-4-methyl-5-(2-nitrooxy-ethyl)-thiazole derivativeshaving a biocleavable moiety, as shown in Tables 1 and 2, have beensimilarly prepared.

TABLE 1 Compound Chemical Structure Pet-2

Pet-3

Pet-4

Pet-5

Pet-6

Pet-7

Pet-8

Pet-9

Pet-10

Pet-11

Pet-12

Pet-13

Pet-14

Pet-15

Pet-16

Pet-17

Pet-18

Pet-19

Pet-20

Pet-21

Pet-22

Pet-23

Pet-24

Pet-25

Pet-26

Pet-27

Pet-28

Pet-29

Pet-30

Pet-31

Pet-32

Pet-33

Pet-34

Pet-35

Pet-36

Pet-37

Pet-38

Pet-39

Pet-40

Pet-41

Pet-42

Pet-43

Pet-44

Pet-45

Pet-46

Pet-47

Pet-48

Pet-49

Pet-50

Pet-51

Pet-52

Pet-53

Pet-54

Pet-55

Pet-56

Pet-57

Pet-58

Pet-59

Pet-60

Pet-61

Pet-62

Pet-63

Pet-64

Pet-65

Pet-66

Pet-67

Pet-68

Pet-69

Pet-70

Pet-71

Pet-72

Pet-73

Pet-74

Pet-75

Pet-76

Pet-77

Pet-78

Pet-79

Pet-80

Pet-81

Pet-82

Pet-83

Pet-84

Pet-85

Pet-86

Pet-87

Pet-88

Pet-89

Pet-90

Pet-91

Pet-92

Pet-93

Pet-94

Pet-95

Pet-96

Pet-97

Pet-98

Pet-99

Pet-100

Pet-101

Pet-102

Pet-103

Pet-104

Pet-105

Pet-106

Pet-107

Pet-108

Pet-109

Pet-110

Pet-111

Pet-112

Pet-113

Pet-114

Pet-115

Pet-116

Pet-117

Pet-118

Pet-119

Pet-120

Pet-121

Pet-122

Pet-123

Pet-124

Pet-125

Pet-126

Pet-127

Pet-128

Pet-129

Pet-130

Pet-131

Pet-132

Pet-133

Pet-134

Pet-135

Pet-136

Pet-137

Pet-138

Pet-139

Pet-140

Pet-141

Pet-142

Pet-143

Pet-144

Pet-145

Pet-146

Pet-147

Pet-148

Pet-149

Pet-150

Pet-151

Pet-152

Pet-153

Pet-154

Pet-155

Pet-156

Pet-157

Pet-158

Pet-159

Pet-160

Pet-161

Pet-162

Pet-163

Pet-164

Pet-167

Pet-168

Pet-169

Pet-170

Pet-171

Pet-172

Pet-173

Pet-174

Pet-175

Pet-176

Pet-177

Pet-178

Pet-179

Pet-180

Pet-181

Pet-182

Pet-183

Pet-184

Pet-185

Pet-186

TABLE 2 Compound Chemical Name Pet-22-Ethyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-32,4-Dimethyl-5-(2-nitrooxy-ethyl)-thiazole Pet-42-Isopropyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-54-Methyl-5-(-2-nitrooxy-ethyl)-2-(1-nitrooxy-ethyl-thiazole Pet-64-Methyl-5-(nitrooxy-ethyl)-2-trifluoromethyl-thiazole Pet-7Dimethyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-84-Methyl-5-(2-nitrooxy-ethyl)-2-phenyl-thiazole Pet-92-Methoxy-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-104-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-ylamine Pet-114-[4-Methyl-5-(2-nitrooxy-ethyl)thiazole-2-yl]-piperidine Pet-123-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-yl]-pyridine Pet-131,4-Bis-[4-Methyl-5-(2-nitrooxy)-ethyl)-thiazol-2-yl]-Butane Pet-142-Benzyloxy-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-151,2-Bis-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-ethane Pet-16Bis-[4-Methyl-5-(2-nitrooxy)-ethyl)-thiazol-2-yl]-methane Pet-172-[1-(6-Methoxy-naphthalen-2-yl-ethyl]-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-182-Chloro-3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-6-trifluoromethyl-pyridine Pet-19Diethyl-{3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridin-4-yl}-amine Pet-202-Methyl-5-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine Pet-213-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine 1-oxide Pet-225-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-2-trifluoromethyl-pyridine Pet-232-Methoxy-6-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]- pyrazinePet-24 Methyl-{6-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyrazin-2-yl}-amine Pet-252-Ethyl-4-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine 1- oxidePet-26 5-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-2-trifluoromethyl-pyridine 1-oxide Pet-272-(2,3-Dichloro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-282-(2,4-Dichloro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-292-(2,6-Dichloro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-302-(3,4-Dichloro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-312-(3,5-Dichloro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-322-(2,3-Difluoro-4-methyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-33 2-(2,6-Difluoro-3-methyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-342-(2,3-Dimethyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-352-(3,4-Dimethyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-362-(2,4-Dichloro-5-fluoro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-37 2-(2-Fluoro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-382-(4-Fluoro-2-methyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-39 2-(2-Fluoro-3-trifluoromethyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-402-(4-Fluoro-2-trifluoromethyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-412-(3-Fluoro-4-trifluoromethyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-42N,N′-Bis-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-hydrazine Pet-43Bis-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-444-Methyl-5-(2-nitrooxy-ethyl)-2-pentafluorophenyl-thiazole Pet-454-Methyl-5-(2-nitrooxy-ethyl)-2-pentafluoroethyl-thiazole Pet-464-Methyl-5-(2-nitrooxy-ethyl)-2-(2-phenyl-propyl)-thiazole Pet-47Hexyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-48N-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-N′-phenyl- hydrazinePet-49 3-Methyl-1-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-butylamine Pet-50N-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-hydroxylamine Pet-51C-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-methylamine Pet-52N-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-formamide Pet-53 Formicacid N′-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]- hydrazide Pet-54Bis-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-disulfide Pet-554-Methyl-5-(2-nitrooxy-ethyl)-2-(2,3,4,5-tetrafluoro-phenyl)- thiazolePet-56 2-(4-Chloro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-575-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-benzene-1,2,3-triolPet-58 4-Methyl-5-(2-nitrooxy-ethyl)-2-(2,4,6-trimethyl-phenyl)-thiazolePet-59 4-Methyl-5-(2-nitrooxy-ethyl)-2-(4-trifluoromethyl-phenyl)-thiazole Pe-604-Methyl-5-(2-nitrooxy-ethyl)-2-(3-trifluoromethyl-phenyl)- thiazolePet-61 4-Methyl-5-(2-nitrooxy-ethyl)-2-(2-trifluoromethyl-phenyl)-thiazole Pet-624-Methyl-5-(2-nitrooxy-ethyl)-2-(2-trifluoromethoxy-phenyl)- thiazolePet-63 4-Methyl-5-(2-nitrooxy-ethyl)-2-(4-trifluoromethoxy-phenyl)-thiazole Pet-64 4-Methyl-5-(2-nitrooxy-ethyl)-2-p-tolyl-thiazole Pet-654-Methyl-5-(2-nitrooxy-ethyl)-2-m-tolyl-thiazole Pet-662-[1-(4-Isobutyl-phenyl)-ethyl]-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-67 2-(4-[1,2]Dithiolan-3-yl-butyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-68 3-(4-Amino-2-methyl-pyrimidin-5-ylmethyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazol-3-ium; chloride Pet-692-Furan-2-yl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-70[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-hydrazine Pet-714-Methyl-5-(2-nitrooxy-ethyl)-2-thiophen-2-yl-thiazole Pet-722-Benzo[b]thiophen-2-yl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-734-Methyl-5-(2-nitrooxy-ethyl)-2-(2,2,5,5-tetramethyl-pyrrolidin-3-yl)-thiazole Pet-744-Methyl-5-(2-nitrooxy-ethyl)-2-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)-thiazole Pet-752,2,5,5-Tetramethyl-3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-2,5-dihydro-pyrrol-1-ol Pet-762-Cyano-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]- acetamide Pet-774-Methyl-5-(2-nitrooxy-ethyl)-2-pyrrolidin-2-yl-thiazole Pet-78[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl-amine Pet-79N-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylmethyl]-acetamide Pet-80[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridin-3-ylmethyl- aminePet-81 4-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine Pet-823-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-piperidine Pet-832-(3,5-Dimethyl-pyrazol-1-yl)-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-84 5-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-1H-imidazol-4-ylamine Pet-85 4-Methyl-5-(2-nitrooxy-ethyl)-2-vinyl-thiazole Pet-864-Methyl-5-(2-nitrooxy-ethyl)-2-thiophen-2-ylmethyl-thiazole Pet-874-Methyl-5-(2-nitrooxy-ethyl)-2-(1-thiophen-2-yl-ethyl)-thiazole Pet-88[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-thiophen-2-yl- methanonePet-89 4-Methyl-5-(2-nitrooxymethoxy-ethyl)-thiazole Pet-902,4-Dimethyl-5-(2-nitrooxymethoxy-ethyl)-thiazole Pet-91[2-(2-Ethyl-4-methyl-thiazol-5-yl)-ethyl]-nitrooxymethyl-amine Pet-922-Isopropyl-4-methyl-5-(2-nitrooxymethylsulfanyl-ethyl)-thiazole Pet-934-Methyl-5-(2-nitrooxy-ethyl)-2-(4-nitrooxy-pyrrolidin-2-yl)- thiazolePet-94 Diethyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-954-Methyl-5-(2-nitrooxy-ethyl)-2-(nitrooxy-thiophen-2-yl-methyl)-thiazole Pet-96N-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-2-nitrooxy- acetamidePet-97 N-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide Pet-98N-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-ylmethyl]-acetamide Pet-994′-Methyl-5′-(2-nitrooxy-ethyl)-2,3,4,5-tetrahydro- [2,2′]bithiazolylPe-100 2-Cyclopropyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-1012-Cyclohexyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-102Bis-[4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-yl]-diazene Pet-1032-Amino-3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propionic acidPet-104 2-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenylaminePet-105 N-{2-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl}-acetamide Pet-1064-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenylamine Pet-107N-{4-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl}- acetamidePet-108 2-Isobutyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-109Diisopropyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-1102,2,5,5-Tetramethyl-3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-2,5-dihydro-pyrrol-1-ol (free radical) Pet-111Dibutyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-112Amino-{3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propylamino}-acetic acid Pet-1134-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acid ethyl esterPet-114 [4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetonitrilePet-115 4,4′-Bis-[4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-yl]-diphenyl-disulfide Pet-116 Acetic acid2-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl ester Pet-1172-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenol Pet-1184,4′-Dimethyl-5,5′-bis-(2-nitrooxy-ethyl)-[2,2′]bithiazolyl Pet-1191-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-2-phenyl- ethylaminePet-120 4-Methyl-5-(2-nitrooxy-ethyl)-2-(4-nitro-phenyl)-thiazolePet-121 2-(4-Ethoxy-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazolePet-122 Diethyl-{4-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl}-amine Pet-123 Dimethyl-{4-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl}-amine Pet-1241-Methyl-4-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]- piperidinePet-125 2-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyrazine Pet-1262-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyrazine 4-oxide Pet-1272-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyrazine 1,4- dioxidePet-128 4-Methyl-2-naphthalen-1-yl-5-(2-nitrooxy-ethyl)-thiazole Pet-1292-(2-Chloro-6-methyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-130 2-(2-Chloro-6-fluoro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-1312-(2-Chloro-4-fluoro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-132 2-(2-Chloro-5-fluoro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-1332-(4-Chloro-2-fluoro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-134 2-(3-Chloro-4-fluoro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-1352-Benzo[1,3]dioxol-5-ylmethyl-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-136 {2-Benzo[1,3]dioxol-5-yl-1-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-ethyl}-methyl-amine Pet-1372,2,4-Trimethyl-6-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-1,2,3,4-tetrahydro-quinoline Pet-1382,2,4-Trimethyl-6-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-3,4-dihydro-2H-quinolin-1-oxide Pet-139trans-2-{4-[2-(3,5-Dimethoxy-phenyl)-vinyl]-phenyl}-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-1404-{4-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-butyl}-tetrahydro-thieno[3,4-d]imidazol-2-one Pet-141(4-{1-[4-Methyl-5-(2-nitrooxy-ethyl)-2-yl]-ethyl}-phenyl)-phenyl-methanone Pet-1422-[1-(2-Fluoro-biphenyl-4-yl0-ethyl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-143(2,6-Dichloro-phenyl)-{2-nitrooxy-ethyl)-thiazol-2-ylmethyl]-phenyl}-amine Pet-1442-Ethyl-5-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine Pet-145(2-Fluoro-phenyl)-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]- aminePet-146 (5-Fluoro-2-methyl-phenyl)-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-147Ethyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-148(3,5-Bis-trifluoromethyl-phenyl)-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-149[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-naphthalen-1-yl- aminePet-150 2-(6-Methoxy-naphthalen-2-yl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propionamide Pet-151 5-[1,2]Dithiolan-3-yl-pentanoic acid[4-methyl-5-(2-nitrooxy- ethyl)-thiazol-2-yl]-amide Pet-1522-(4-Isobutyl-phenyl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-propionamide Pet-1534-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acid N′-phthalazin-1-yl-hydrazide Pet-154N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-nicotinamide Pet-155allyl-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine Pet-156N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-1-oxy- nicotinamidePet-157 4-acetylamino-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-benzamide Pet-158 hexadecanoic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]- amide Pet-159 acetic acid2-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2- ylcarbamoyl]-phenyl esterPet-160 pyrrolidine-2-carboxylic acid [4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amide Pet-1612,6-difluoro-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]- benzamidePet-1622-(2,4-dichloro-phenyl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide Pet-1632-(2,4-dichloro-phenoxy)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide Pet-1644-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acid 10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl ester Pet-1651-[4-Methyl-5-(2-nitrooxy-ethyl)thiazol-2-ylmethyl]-pyrrrolidin- 2-onePet-166 2-Propyl-pentanoic acid[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2- yl]-amide Pet-1674-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acid Pet-1684-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acid ethyl esterPet-169 4-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acid phenylester Pet-170 4-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acidpyridin- 3-ylamide Pet-1715-Dibenzylamino-2-hydroxy-N-[4-methyl-5-(-2-nitrooxy-ethyl)-thiazol-2-yl]-benzamide Pet-1723-[4-Methyl-5-(2-nitrooxy-ethyl)thiazol-2-ylmethyl]-1H-indole Pet-1732-{1-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-ethylamino}-4-phenyl-butyric acid ethyl ester Pet-174[4-Methyl-5-(2-nitrooxy-ethyl)-2-yl]-pyridin-4-yl-amine Pet-1752-(4-[1,3]Dithiolan-2-yl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-176 2-(4-[1,3]Dioxolan-2-yl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-1772-(4-tert-Butyl-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-1784-{4-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl}-[1,2,3]thiadiazole Pet-1792-Hydroxy-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazole-2-yl]-5-[4-(pyridine-2-ylsulfamoyl)-phenylazo]-benzamide Pet-1802-(1-Aminomethyl-cyclohexyl)-N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-acetamide Pet-1812-(3,5-difluoro-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole Pet-1822-(5-chloro-2-methoxy-phenyl)-4-methyl-5-(2-nitrooxy-ethyl)- thiazolePet-183 N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-guanidine Pet-184N-(2-chloro-benzylidene)-N′-[4-methyl-5-(nitrooxy-ethyl)-thiazol-2-yl]-hydrazine Pet-185(4-chlorophenyl)-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]- aminePet-186 (3,5-dichloro-phenyl)-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-amine

In Vitro Studies

Experimental Methods:

In Vitro Vasorelaxation:

In order to measure the vasorelaxation effect in vitro, two parameterswere followed in the assays: the mechanical contraction and relaxationof aortae tissue before and during dose response exposure of aortaetissues to the compounds of the present invention as well as to thecontrol compound GTN, and the quantitative levels of c-GMP before andafter exposing aortae tissues to the compounds of the present inventionas well as to the control compound GTN.

Male Sprague Dawley rats were anaesthetized using intraperitonealinjection of ketamine and xylazine (50 and 10 milligram per kilogram,respectively) and the thoracic aorta, the large upper portion of thedescending aorta, starting at the caudal border of the chest bone, wasremoved thereafter, while carefully removing the paraadventetial tissuesurrounding the vessel. Aortae were cut into rings of 4-5 millimetersand mounted onto the tissue path. The path buffer (Krebs-bicrbonate) wasconstantly gassed with carbogen and maintained at 37° C. The rings werepreloaded under 2 g tension and equilibrated for 90 minutes, whilerefreshing the buffer every 15 minutes. After stabilization, the ringswere contracted with epinephrine (1 μM). CumulativeConcentration-Response Curves (CCRC) were measured for the vasorelaxingeffect induced by adding various concentrations, ranging from 10⁻¹⁰ M to10⁻⁵ M of Pet-2, Pet-3, Pet-7, Pet-8, Pet-12, Pet-24, Pet-43, Pet-59,Pet-147, Pet-152 and Pet-154, and glyceryltrinitrate (GTN), according tothe procedure described in Feelich et al., Molecular Pharmacology,56:243-253 (1999). The vehicle, which was used for this experiment waswater:ethanol:propyleneglycol at proportional ratio of 1:1:1.

In Vitro Induction of Tolerance to NO Releasing Compounds:

Aortic rings were prepared as described hereinabove, and were exposed to0.44 mM GTN (vehicle was water:ethanol:propyleneglycol at proportionalratio of 1:1:1) for 1 hour before CCRC construction, to thereby inducetolerance to nitroglycerine. At the end of the tolerance inductionperiod, rings were thrice washed with the path buffer (Krebs-bicrbonate)every 15 minutes for the following hour and then CCRC for vasorelaxationon GTN-induced tolerance rings were constructed for GTN (pretreatedversus untreated rings, FIG. 15), in order to verify the ability toinduce and measure GTN tolerance induction in vitro

Attempted induction of tolerance to the NO-donating compounds of thepresent invention was performed under the same conditions as for GTN.Thus, in an exemplary experiment, rings were exposed to 0.44 mM solutionof Pet-3 in the attempt to induce tolerance for Pet-3. CCRC were thenmeasured for the vasorelaxing effect of Pet-3 on a group of rings whichwere pre-exposed to Pet-3 as to induce tolerance to Pet-3, and for anuntreated control group. The above protocol was repeated with Pet-2,Pet-7, Pet-8, Pet-12, Pet-24, Pet-43, Pet-149 and Pet-152.

Cross-tolerance studies were conducted using the same techniquedescribed above for measuring vasorelaxation of aortic rings from rats.Two groups of aortic rings samples were pre-treated with GTN, then onegroup was measured for vasorelaxation by GTN and the second group forvasorelaxation by Pet-12, an exemplary NO-donor according to the presentinvention. For the opposite cross-tolerance studies, two groups ofaortic rings samples were pre-treated with Pet-12, then one group wasmeasured for vasorelaxation by GTN and the second group forvasorelaxation by Pet-12.

Cyclic-GMP Measurement in Aortic Rat Tissue:

To determine the c-GMP-producing effect of some exemplary NO-donorsaccording to the present invention, as compared with that of GTN, ringswere treated as described above in the vasorelaxation effect studiesexcept that they were not hooked to the transducer. GTN (10⁻⁶ M) wasadded to the chamber and, 1 minute after exposure to the drug, rings(usually 3-5) were taken, wrapped with aluminum foil and immediatelyfrozen in liquid nitrogen for storage. Control sample group was treatedwith the vehicle solution only. At the time of analysis, rings werethawed, blotted over a dry gauzed and weighted. A known weight of tissuewas homogenized in 2 ml ice-cold modified Hank's balanced salt solutioncontaining (in gram/liter) NaCl 8; KCl 0.4; glucose 1; KH₂PO₄ 0.06;Na₂HPO₄ 0.047; Phenol red 0.017 and further containing 25 mM EDTA(disodium salt). The homogenate was centrifuged at 4000 g (force units)for 10 minutes at 2-4° C. and the supernatant was thereafter transferredinto fresh pre-cooled test tube containing 1 ml of acetonitrile. Thetubes were vortex mixed for a few seconds and subsequently centrifugedat 4000 g for 5 minutes at 2-4° C., to remove the precipitated protein.Each supernatant was transferred into a clean test tube and evaporatedto dryness under a stream of nitrogen at 55° C. The dry residue wasreconstituted with 10 volumes of Tris-EDTA buffer pH 7.5 (0.05 M Triscontaining 4 mM EDTA). Aliquots of 100 μl of the reconstituted solutionin duplicate were used for c-GMP measurements using Amersham RIA kit, asis described in Haj-Yehia & Benet [Pharmacology, 1995, 273, 1, pp.94-100]. Standard curves were plotted with six concentrations (0, 0.5,1, 2, 4 and 8 pmoles of c-GMP per tube).

Experimental Results:

In Vitro Vasorelaxation:

The vasorelaxing effect of the thiazole-derived NO-donors according tothe present invention was measured on slices of thoracic aorta of ratscontracted with epinephrine, as described hereinabove.

The obtained results for Pet-2, Pet-3, Pet-7, Pet-8, Pet-12, Pet-24,Pet-43, Pet-59, Pet-147, Pet-152 and Pet-154 are presented in FIGS.2-14, and are summarized in Table 3 below. The results clearly show thatthe thiazole-based NO-donors according to a preferred embodiment of thepresent invention are highly active in inducing a vasorelaxation effectand are highly superior in this respect to GTN, being 10-fold moreactive than GTN in exemplary cases.

TABLE 3 Compound Concentration EC₅₀ Figure. No. GTN 4.47E−08 −7.55 3 to6 and 8 to 14 Pet-2 2.51E−08 −7.6 2 and 3 Pet-3 3.55E−08 −7.45 4 Pet-72.82E−08 −7.55 5 Pet-8 2.82E−08 −7.7 6 Pet-12 3.98E−08 −7.4 7 Pet-242.00E−07 −6.7 8 Pet-43 1.59E−07 −6.8 9 Pet-59 7.94E−08 −7.1 10 Pet-1473.16E−08 −7.5 11 Pet-152 5.01E−08 −7.3 12 Pet-154 3.98E−08 −7.4 13

In Vitro Induction of Tolerance to NO Releasing Compounds:

The absence of tolerance induction by the thiazole-based NO-donorsaccording to the present invention was demonstrated by pre-exposingslices of thoracic aorta of rats to the test compounds followed byvasorelaxation measurements as described hereinabove.

The obtained results for Pet-2, Pet-3, Pet-7, Pet-8, Pet-12, Pet-24,Pet-43, Pet-149 and Pet-152 are presented Table 4 below and are furthershown in FIGS. 15-23. These results clearly demonstrate that theNO-donors of the present invention do not induce tolerance and are thushighly superior to GTN.

The results obtained in the cross-tolerance studies described above arepresented in FIGS. 19 and 20 and clearly show that the induced toleranceeffect to GTN has no effect on the vasorelaxation capacity of Pet-12, anexemplary NO-donor according to the present invention (FIG. 19), andthat Pet-12 does not evoke tolerance to itself not to GTN. The resultsobtained in these (FIG. 20).

TABLE 4 Compound Concentration EC₅₀ Figure. No. GTN 4.47E−08 −7.5 15Pet-2 3.98E−08 −7.4  3 Pet-3 5.01E−08 −7.3 16 Pet-7 5.01E−08 −7.45 17Pet-8 2.51E−08 −7.6  6 Pet-12 6.31E−08 −7.2 18 to 20 Pet-24 2.51E−07−6.6 21 Pet-43 2.51E−07 −6.6 22 Pet-149 4.33E−08 −7.4 23 Pet-1525.01E−08 −7.3 33 Pet-154 3.98E−08 −7.4 34 Pet-155 5.01E−08 −7.3 32

Cyclic-GMP Measurement in Aortic Rat Tissue:

The NO-tolerance effect of GTN and NO-donor compounds according to thepresent invention was measured by following the production of c-GMP inslices of thoracic aorta of rats. Thus, the production of c-GMP wasmeasured as described in the methods section above for the referenceNO-donor compound GTN, and for the NO-donors of the present inventionPet-2, Pet-3, Pet-7, Pet-8, Pet-10, Pet-12, Pet-24, Pet-59, Pet-147,Pet-149, Pet-152, Pet-154 and Pet-155.

The results are summarized in Table 5 hereinbelow and clearly show thatwhile the c-GMP level was significantly reduced following a repeatedtreatment with GTN, it remained almost unchanged following a repeatedtreatment with each of the tested NO-donors of the present invention,thus the lack of tolerance induction thereby. These obtained results arefurther presented in FIG. 25, for Pet-3, Pet-7 and Pet-8, FIG. 27 forPet-24 and Pet-149, and FIG. 33 for Pet-10, Pet-59, Pet-147, Pet-152,Pet-154 and Pet-155, all of which show the increased levels of cGMP whentreated with the exemplary NO-donating compound of the presentinvention, as compared with GTN.

TABLE 5 cGMP (pmol/g tissue) Tested Compound Pre-infusion Post-infusionGTN 127 ± 11  45 ± 12^(¥) Pet-2 143 ± 13 140 ± 13^(§) Pet-3 122 ± 14 118± 10^(§) Pet-7 120 ± 12 123 ± 12^(§) Pet-8 133 ± 10 136 ± 12^(§) Pet-12127 ± 11 112 ± 12^(§) Pet-24 115 ± 14 112 ± 10^(§) Pet-149 112 ± 12 115± 14^(§) ^(¥)Significantly different from pre-infusion values anddenotes tolerance ^(§)Not significantly different from pre-infusionvalues and denotes the lack of tolerance

In Vitro Studies

Experimental Methods:

In order to measure the vasorelaxation effect in vivo, two parameterswere observed: the mechanical contraction and relaxation of aortaetissue before and during exposure of aortae tissues to the compounds ofthe present invention as well as to the reference compound GTN, and thequantitative levels of c-GMP before and after exposing aortae tissues tothe compounds of the present invention as well as to the referencecompound GTN. These techniques were also used to detect toleranceinduction by the NO-donors of the present invention and by GTN.

For c-GMP levels and tolerance development, the rat's thoracic aorta wasisolated after exposure to the tested compound in vivo, and measured asdescribed above in the in vitro assays.

Hypotensive Effects:

Mean arterial pressure (MAP) was monitored and determined during bolusinfusion of Pet-2, Pet-3, Pet-7, Pet-8, Pet-12 and Pet-13. Briefly,polyethylene catheters (PE50) were inserted into the carotid artery andinto the jugular vein of anesthetized hypertensive Sprague-Dawley malerats (200-300 grams). A blood pressure recorder was placed within thecarotid artery. Phenylephrine (0.1 mg) was then injected and the meanarterial blood pressure (MABP) was monitored continuously until a stablebaseline was reached. The hypotensive effects of intravenous (iv) bolusdoses (0.1 mg of each tested compound) administered intravenously at thejugular vein in minimal volume of vehicle (0.2 ml per dose) wereevaluated. Five minutes were allowed between each dose, during which themaximal decrease in MAP was recorded and a dose-response graph comparingthe hypotensive effect of the compounds was constructed.

Induction of In Vivo Nitrate Tolerance:

Male Sprague Dawley rats (220 to 250 grams) were housed under constanttemperature and humidity conditions. Light was maintained at a 12/12light dark cycle. Except for 12 hours before the experiment, when foodfasting was initiated, all rats had free access to standard rat pelletdiet and tap water. The animals were anesthetized by intraperitonealinjection of ketamine and xylazine combination (50 and 10 milligram perkilogram, respectively). Chronic catheterization of the superior venacava was performed as described by Boesgaard et al. [Circ Res. 1994, 74,115-120], and two catheters (medical-grade Tygon catheters) with thetips in the right atrium were implanted through a transverse incision atthe mid portion of the neck via the left and right external jugularveins. The catheters were filled with normal saline solution containing1000 IU/ml of heparin and plugged with a metal pin. Catheters wereexternalized to the back of the neck region and secured by a polyesterfelt disk.

After implantation, the rats were individually housed until theyregained their preoperative weight and appeared healthy (3 to 4 dayspost-operation). At this time point (day 0), an osmotic mini-pump (AlzaCorp., Palo Alto, Calif.) delivering constant volume (10 μl/hour) ofvehicle or drug was connected to one of the intravenous catheters.

For inducing tolerance to the tested NO-donor according to the presentinvention, the animals received 200 μg/hour of the tested compound for16 hours (short-term-induced tolerance). A similar number of animalswere processed by the same surgical procedure and received the vehiclealone (10 μl/hour of a solution consisting of 30% ethanol and 30%propylene glycol in sterile distilled water for injection).

At the end of the infusion periods used for the development of tolerance(16 hour), the rats were anesthetized as described above, the twocatheters were externalized and the delivering catheters weredisconnected from the mini-pump. The abdomen was opened by midlineincision and the thoracic aorta were isolated, washed and cleaned fromthe surrounding tissues. Strips of the treated aortas were pooled inchambers containing Krebs-Hanseliet buffer (10 ml), and exposed to 1 μMof the tested NO-donor (final concentration) for 120 seconds. Thetissues were quickly collected and frozen in liquid nitrogen for cGMPmeasurement, described hereinabove.

Experimental Results:

Hypotensive effects:

The results obtained from evaluating the hypotensive effects of Pet-2,Pet-3, Pet-7, Pet-8, Pet-12, Pet-13 and GTN in vivo, are presented inFIGS. 28-31. These results clearly demonstrate the high activity of thecompound of the present invention as hypotensive agents. Unlike GTN, inwhich attenuation in its effect on MAP was observed, the hypotensiveeffect of the NO-donating compounds of the present invention on MAP wasconsistent and reproducible following successive administrations of thecompounds, thus indicating the prevention of tolerance development bythese exemplary compounds.

Induction of In Vivo Nitrate Tolerance:

The in vivo nitrate tolerance was measured, as described hereinabove,for GTN, Pet-2 and Pet-12. The results obtained are presented in FIG.24, which shows the absence of tolerance effect induced by Pet-2 ascompared with GTN, and in FIG. 26, which shows the absence of toleranceeffect induced by Pet-12.

As is shown in FIG. 24, upon exposure to GTN, the level of cGMPfollowing a repeated treatment with GTN was decreased, thus indicatingtolerance induction, whereby upon exposure to Pet-2, the levels of c-GMPremained substantially unchanged following a repeated treatment withPet-2. As is shown in FIG. 26, the same lack of tolerance induction wasobserved with repeated treatment with Pet-12. These results clearlydemonstrate that Pet-2 and Pet-12, two exemplary NO-donor compoundsaccording to the present invention, are highly superior to GTN by beinga non-tolerance inducing NO-donating compound.

The results presented herein clearly demonstrate that the NO-releasingcapacity of the NO-donors according to the present invention, measuredas a capacity to include vasorelaxation both in vitro and in vivo, issuperior to that of GTN. The results further demonstrate that theNO-donors of the present invention do not induce tolerance both in vitroand in vivo, nor is their capacity to exert vasorelaxation affected byinduced tolerance to GTN.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

1. An NO-donating compound having the general formula I:

wherein: A is selected from the group consisting of alkyl, amine, aryl,C-amide, carbonyl, hydrazine, N-amide and any combination thereof, orabsent; X is a heteroaryl selected from the group consisting ofbenzodioxole, benzothiophene, diazole, dithiolane, furan, imidazole,indole, phthalazine, piperidine, pyrazine, pyrazole, pyridine,pyridinyl, pyrimidine, pyrrolidine, quinoline and thiadiazole; B is anethylene chain; Y is —ONO₂; and Z is methyl.
 2. The NO-donating compoundof claim 1, wherein said heteroaryl is pyridine.
 3. An NO-donatingcompound selected from the group consisting of:3-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-yl]-pyridine (Pet-12);2-Chloro-3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-6-trifluoromethyl-pyridine(Pet-18);Diethyl-{3-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridin-4-yl}-amine(Pet-19);2-Methyl-5-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine(Pet-20); 3-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine1-oxide (Pet-21);5-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-2-trifluoromethyl-pyridine(Pet-22);2-Methoxy-6-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyrazine(Pet-23);Methyl-{6-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyrazin-2-yl}-amine(Pet-24);2-Ethyl-4-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine 1-oxide(Pet-25);5-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-2-trifluoromethyl-pyridine1-oxide (Pet-26);3-(4-Amino-2-methyl-pyrimidin-5-ylmethyl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazol-3-ium;chloride (Pet-68); 2-Furan-2-yl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-69); 4-Methyl-5-(2-nitrooxy-ethyl)-2-thiophen-2-yl-thiazole(Pet-71); 2-Benzo[b]thiophen-2-yl-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-72);[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridin-3-ylmethyl-amine(Pet-80); 4-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine(Pet-81);2-(3,5-Dimethyl-pyrazol-1-yl)-4-methyl-5-(2-nitrooxy-ethyl)-thiazole(Pet-83);5-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-1H-imidazol-4-ylamine(Pet-84); 4-Methyl-5-(2-nitrooxy-ethyl)-2-thiophen-2-ylmethyl-thiazole(Pet-86);4-Methyl-5-(2-nitrooxy-ethyl)-2-(1-thiophen-2-yl-ethyl)-thiazole(Pet-87);[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-thiophen-2-yl-methanone(Pet-88);4-Methyl-5-(2-nitrooxy-ethyl)-2-(nitrooxy-thiophen-2-yl-methyl)-thiazole(Pet-95); 2-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyrazine(Pet-125); 2-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyrazine4-oxide (Pet-126);2-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyrazine 1,4-dioxide(Pet-127);2-Ethyl-5-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-pyridine(Pet-144); 4-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acidN′-phthalazin-1-yl-hydrazide (Pet-153);N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-nicotinamide (Pet-154);N-[4-methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-1-oxy-nicotinamide(Pet-156); 4-Methyl-5-(2-nitrooxy-ethyl)-thiazole-2-carboxylic acidpyridin-3-ylamide (Pet-170);3-[4-Methyl-5-(2-nitrooxy-ethyl)thiazol-2-ylmethyl]-1H-indole (Pet-172);[4-Methyl-5-(2-nitrooxy-ethyl)-2-yl]-pyridin-4-yl-amine (Pet-174); and4-{4-[4-Methyl-5-(2-nitrooxy-ethyl)-thiazol-2-yl]-phenyl}-[1,2,3]thiadiazole(Pet-178).
 4. A pharmaceutical composition comprising, as an activeingredient, the NO-donating compound of claim 1 and a pharmaceuticallyacceptable carrier.
 5. A medical device comprising the NO-donatingcompound of claim 1 and a delivery system configured for delivering saidNO-donating compound to a bodily site of a subject.
 6. The medicaldevice of claim 5, wherein said NO-donating compound forms a part of apharmaceutical composition, said pharmaceutical composition furthercomprising a pharmaceutically acceptable carrier.
 7. The medical deviceof claim 5, wherein said delivering is effected by inhalation.
 8. Themedical device of claim 7, wherein said delivery system is selected fromthe group consisting of a metered dose inhaler, a respirator, anebulizer inhaler, a dry powder inhaler, an electric warmer, avaporizer, an atomizer and an aerosol generator.
 9. The medical deviceof claim 5, wherein said delivering is effected transdermally.
 10. Themedical device of claim 9, wherein said delivery system is selected fromthe group consisting of an adhesive plaster and a skin patch.
 11. Themedical device of claim 5, wherein said delivering is effectedtopically.
 12. The medical device of claim 11, wherein said deliverysystem is selected from the group consisting of an adhesive strip, abandage, an adhesive plaster, a wound dressing and a skin patch.
 13. Themedical device of claim 5, wherein said delivering is effected byimplanting the medical device in a bodily organ.
 14. The medical deviceof claim 13, wherein said delivery system is selected from the groupconsisting of an aortic aneurysm graft device, an atrioventricularshunt, a catheter, a defibrilator, a heart valve, a hemodialysiscatheter, a hemodialysis graft, an indwelling arterial catheter, anindwelling venous catheter, a needle, a pacemaker, a pacemaker lead, apatent foramen ovale septal closure device, a stent, a stent graft, asuture, a synthetic vascular graft, a thread, a tube, a vascularanastomosis clip, a vascular aneurysm occluder, a vascular clip, avascular prosthetic filter, a vascular sheath and a drug delivery port,a venous valve and a wire.
 15. The medical device of claim 13, whereinsaid organ is selected from the group consisting of a pulmonary cavity,a blood vessel, an artery, a vein, a capillary, a heart, a heart cavityand a visceral organ.
 16. A pharmaceutical composition comprising, as anactive ingredient, the NO-donating compound of claim 3 and apharmaceutically acceptable carrier.
 17. A medical device comprising theNO-donating compound of claim 3 and a delivery system configured fordelivering said NO-donating compound to a bodily site of a subject. 18.The medical device of claim 17, wherein said NO-donating compound formsa part of a pharmaceutical composition, said pharmaceutical compositionfurther comprising a pharmaceutically acceptable carrier.
 19. Themedical device of claim 17, wherein said delivering is effected byinhalation.
 20. The medical device of claim 19, wherein said deliverysystem is selected from the group consisting of a metered dose inhaler,a respirator, a nebulizer inhaler, a dry powder inhaler, an electricwarmer, a vaporizer, an atomizer and an aerosol generator.
 21. Themedical device of claim 17, wherein said delivering is effectedtransdermally.
 22. The medical device of claim 17, wherein saiddelivering is effected topically.
 23. The medical device of claim 17,wherein said delivering is effected by implanting the medical device ina bodily organ.